Uppala Radhakrishna ^1* Rupa Radhakrishnan ² Lavanya V. Uppala ³ Srinivas Muvvala ⁴ Jignesh Prajapati ⁵ Rakesh Rawal ⁵ Ray O. Bahado-Singh ⁶ Senthilkumar Sadhasivam ¹

• ¹ University of Pittsburgh, Pittsburgh, Pennsylvania, United States
• ² School of Medicine, Indiana University Bloomington, Indianapolis, Indiana, United States
• ³ University of Nebraska Omaha, Omaha, Nebraska, United States
• ⁴ Yale University, New Haven, Connecticut, United States
• ⁵ Gujarat University, Ahmedabad, Gujarat, India
• ⁶ Beaumont Health, Royal Oak, United States

Background: Neonatal Opioid Withdrawal Syndrome (NOWS) is a consequence of inutero exposure to prenatal maternal opioids, (OUD), resulting in the manifestation of symptoms like irritability, feeding problems, tremors, and withdrawal signs. Opioid use during pregnancy can significantly affectprofoundly impact both the mother and the fetus, potentially causing disruptions in normal disrupting fetal brain neurotransmission in the fetal brain and possibly triggering and potentially leading to long-term neurological or, behavioral effects in the child's later life., and vision issues, and increased infant mortality.Drug resistance complicates OUD and NOWS treatment for opioid use disorders (OUD) and NOWS, with protein kinase regulation of drug transporters in these conditions still not fully understood. A better understanding of prenatal opioid exposure-related epigenetic changes to protein kinases and drug transporters could help optimize the management of OUD during pregnancy and NOWS, and minimize the risks of maternal relapse and severe NOWS requiring longer hospitalization.Methods:DNA methylation levels of ATP-binding cassette (ABC) and solute carrier (SLC) drug transporters, along with protein kinase C (PKC) genes, were evaluated in 96 placental samples using the Illumina Infinium MethylationEPIC array BeadChip (850K) assay. Placental tissue samples). Samples were collected from three distinct groups: 32 mothers with infants prenatally exposed to opioids who needed pharmacological intervention for NOWS, 32 mothers with prenatally opioid-exposed infants who did not necessitate NOWS treatment, and 32 mothers who were not exposed to opioids during pregnancy.We identified statistically significant (FDR p-value < 0.05) methylation changes in 87 drug transporters and protein kinases. Among 69 SLC transporters, 24 are significantly differentially methylated SLCs, with 24 hypermethylated, and 34 are hypomethylated, and 11 exhibitexhibiting both hypo-types of methylation changes including SLC13A3, SLC15A2, SLC16A11, SLC16A3, SLC19A2, and hypermethylation. Furthermore, we detectedSLC26A1. We identified methylation changes in 11 ABC drug transporters: (ABCA1, ABCA12, ABCA2, ABCB10, ABCB5, ABCC12, ABCC2, ABCC9, ABCE1, ABCC7, ABCB3): 3 showed hypermethylation, 3 displayed hypomethylation, and 5 exhibited both hyper-and hypomethylation at CpG sites associated with ABCs.Moreover, methylation changes were detected in. Additionally, 7 PKC family genes:(PRKCQ, PRKAA1, PRKCA, PRKCB, PRKCH, PRKCI, and PRKCZ. The) showed methylation changes. These genes are associated with 13 pathways involved in NOWS,