Methadone has been reported to downregulate the expression of glutathione S-transferase P1 (GSTP1) among nine antioxidant genes in SH-SY5Y cells after both short- and long-term treatment. GSTP1 plays a key role in the detoxification of many xenobiotics and is frequently associated with various diseases, especially tumors. The objective of this study is to determine whether this change is reversible. Two different treatment protocols were used. The first protocol evaluated the reversibility of the GSTP1 mRNA change, while the second protocol evaluated the methylation status of the GSTP1 promoter site. To investigate the reversibility of the GSTP1 mRNA change, SH-SY5Y cells were treated with methadone. The drug was then removed from the medium and the cells were cultured in methadone-free medium for a period of time. GSTP1 mRNA levels were expressed as cycle threshold (Ct) values using TATA box-binding protein as a calibrator gene. Methylation at the promoter site was detected by bisulfite treatment. The analysis of variance revealed no significant change in GSTP1 mRNA levels in the cells after methadone was removed from the medium of methadone-treated cells. The study also examined the methylation status of a CpG island in the promoter of GSTP1 in the treated cells. The results demonstrate that although methadone downregulates the mRNA level of GSTP1 in treated cells, it does not induce methylation in the GSTP1 promoter region. The expression of the GSTP1 remains downregulated even after methadone removal from SH-SY5Y cell culture medium; however, methylation of the GSTP1 promoter site does not play a role in this process.

中文翻译：

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美沙酮诱导的 SH-SY5Y 细胞中不可逆的 GSTP1 下调

据报道，在短期和长期治疗后，美沙酮可下调 SH-SY5Y 细胞中九种抗氧化基因中谷胱甘肽 S-转移酶 P1 (GSTP1) 的表达。GSTP1 在许多外源物质的解毒中发挥着关键作用，并且经常与多种疾病，尤其是肿瘤相关。本研究的目的是确定这种变化是否可逆。使用了两种不同的治疗方案。第一个方案评估 GSTP1 mRNA 变化的可逆性，而第二个方案评估 GSTP1 启动子位点的甲基化状态。为了研究 GSTP1 mRNA 变化的可逆性，用美沙酮处理 SH-SY5Y 细胞。然后从培养基中除去药物，并将细胞在不含美沙酮的培养基中培养一段时间。使用 TATA 盒结合蛋白作为校准基因，将 GSTP1 mRNA 水平表示为循环阈值 (Ct)。通过亚硫酸氢盐处理检测启动子位点的甲基化。方差分析显示，从美沙酮处理的细胞培养基中去除美沙酮后，细胞中 GSTP1 mRNA 水平没有显着变化。该研究还检测了处理细胞中 GSTP1 启动子中 CpG 岛的甲基化状态。结果表明，虽然美沙酮下调处理细胞中 GSTP1 的 mRNA 水平，但它不会诱导 GSTP1 启动子区域甲基化。即使从 SH-SY5Y 细胞培养基中去除美沙酮后，GSTP1 的表达仍然下调；然而，GSTP1 启动子位点的甲基化在此过程中不起作用。