P06-21 MEHP treatment induces a decrease in SIRT1 expression, mitochondrial dysfunction, and transcriptomic alterations in the human trophoblastic cell line HTR-8/svneo

ABSTRACT

Background: Mono(ethylhexyl) phthalate (MEHP) is the main metabolite of Di(2-ethylhexyl) phthalate (DEHP), a chemical worldwide used as a plastic softener to increase the malleability, flexibility, and durability of several types of plastic, including those employed in bottled water, medical devices, and food wrapping, among others. Importantly, the consumption of these products has dramatically increased during the COVID-19 pandemic. MEHP has been classified as an endocrine disruptor chemical (EDC) and its involuntary intake has been associated with pregnancy complications such as preeclampsia and miscarriages. The placenta is a transitory organ that provides sustainability to the fetus, as well as the transportation of nutrients, hormones, and oxygen. Recent studies have proposed that MEHP may impair placental development and functionality. Nevertheless, little is known about its molecular mechanisms and effects on the placenta. Recent data has suggested that Sirtuin 1 (SIRT1) might be a molecular target. The aim of this study was to analyze the toxic and transcriptomic effects of MEHP in the human trophoblastic cell line HTR-8/svneo, focusing on the SIRT1-related pathways. Methods and Results: The HTR-8/svneo cell line was used as an extravillous trophoblast model to investigate MEHP effects. MEHP concentrations employed in this study were 0.5, 5, 50, 100, and 200 microM. Cell viability was evaluated by two methods: fixable viability staining, using eFluor 780, and MTT assay. Only the MTT assay suggested a significant decrease in cell viability at 48 hours with MEHP treatments of 5, 50, 100, and 200 microM. Mitochondrial biogenesis was analyzed by qPCR amplifying a region of the MT-ND1 mitochondrial gene. GAPDH promoter was used as a reference control. The results suggested a decrease in mitochondrial DNA at 48 hours. The transcriptomic analysis was performed in an Illumina Next-seq 500 with a coverage of 10 million reads. Doses of 5 and 200 microM of MEHP at 48 hours were analyzed. The results show that 41 and 341 genes were differentially expressed, respectively. These genes are involved in trophoblast function and pathophysiology and, according to previous reports, a portion of them are regulated by SIRT1. Finally, the effect of MEHP on SIRT1 was explored at both protein and mRNA levels by western blot and RT-qPCR, respectively. The results for mRNA levels exhibited a significant decline at 24 hours for all treatments, while protein levels were significantly reduced by 200 microM MEHP treatment at 48 hours. Conclusion(s) The present study demonstrates that MEHP treatments promote mitochondrial dysfunction in HTR-8/svneo cells. Moreover, the transcriptome analysis showed that MEHP modifies important signals for placental function and pathophysiology. The decline in SIRT1 levels correlates with the mitochondrial alterations as well as a portion of the transcriptomic changes, suggesting that SIRT1 may have an important role in MEHP effects in trophoblastic cells. Copyright © 2022 Elsevier B.V.