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通讯作者:

王晓伟,E-mail:wangxiaowei@njmu.edu.cn

中图分类号:R329.28

文献标识码:A

文章编号:1007-4368(2022)11-1507-08

DOI:10.7655/NYDXBNS20221102

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目录contents

    摘要

    目的:探究邻苯二甲酸二(2-乙基己基)酯[di(2-ethylhexyl)phthalate,DEHP]暴露对PI3K/AKT信号通路和人冠状动脉内皮细胞(human coronary artery endothelial cell,HCAEC)的影响,探索DEHP暴露致HCAEC损伤的机制。方法:根据相关文献研究,用不同浓度DEHP处理HCAEC,设置对照组(DMSO)和实验组(DEHP 128、256、384、512 μmol/L)。用CCK-8法测定细胞增殖能力;体外成管实验评估细胞成管能力;流式细胞术评估细胞凋亡水平;Western blot 检测细胞中 p-PI3K、 PI3K、p-AKT、AKT和凋亡相关蛋白BAX、Bcl-2、cleaved Caspase-3、Caspase-3的水平。结果:CCK-8结果显示,DEHP以浓度和时间依赖的方式抑制HCAEC的增殖(P < 0.05)。体外成管实验结果显示,实验组细胞形态多呈孤立的圆形、卵圆形,当DEHP 浓度≥256 μmol/L时,其形成交点数目较对照组减少(P < 0.05)。流式细胞实验结果显示,实验组凋亡比例较对照组上升(P < 0.05)。Western blot结果显示实验组Bcl-2表达水平较对照组降低,同时BAX和cleaved Caspase-3/Caspase-3的水平升高(P < 0.05);当DEHP≥256 μmol/L时,p-PI3K/PI3K、p-AKT/AKT比值较对照组降低(P < 0.05)。结论:DEHP可通过抑制PI3K/AKT/信号通路诱导HCAEC凋亡,抑制其增殖、血管形成能力。

    Abstract

    Objective:This study aims to investigate the effects of di(2-ethylhexyl)phthalate(DEHP)exposure on PI3K/AKT signal pathway and human coronary artery endothelial cells(HCAECs),and to provide a theoretical basis for the treatment of HCAECs damaged by DEHP exposure. Methods:According to relevant literature research,HCAECs were treated with different concentrations of DEHP and divided into control group(DMSO)and experimental group(DEHP 128,256,384,512 μmol/L). The ability of cell proliferation was measured by CCK- 8 assay,the ability of cell tube formation was evaluated by in vitro tube formation assay,the level of apoptosis was evaluated by flow cytometry,and the levels of p-PI3K,PI3K,p-AKT,AKT and apoptosis-related proteins BAX,Bcl-2, cleaved Caspase-3 and Caspase-3 were detected by Western blot. Results:CCK-8 assay showed that DEHP inhibited the proliferation ability of HCAECs in a concentration- and time-dependent manner(P < 0.05). The results of in vitro tube formation assay showed that the morphology of cells in the experimental group was mostly isolated round and oval. The number of intersections formed was reduced compared with the control group(P < 0.05)when the DEHP concentration was ≥256 μmol/L. The results of flow cytometry showed that the proportion of apoptosis in the experimental group was higher than that in the control group(P < 0.05). Western blot results showed that the expression level of Bcl - 2 in the experimental group was lower than that in the control group,while the levels of BAX and cleaved Caspase-3/Caspase-3 increased(P < 0.05). And the ratio of p-PI3K/PI3K and p-AKT/AKT were lower than those in the control group(P < 0.05)when DEHP≥256 μmol/L. Conclusion:DEHP can induce apoptosis and inhibit the proliferation and angiogenesis of HCAECs by inhibiting PI3K/AKT/ signal pathway.

  • 邻苯二甲酸二(2⁃乙基己基)酯[di(2⁃ethylhex⁃ yl)phthalate,DEHP]是一种邻苯二甲酸酯类,因其弹性高、可塑性强,常被用作增塑剂添加于各种聚氯乙烯产品中,如医疗器械、药品和食品容器、儿童玩具和工业塑料等,以提高产品的柔韧性和耐久性[1]。 DEHP以非共价化学键与聚氯乙烯结合,在温度、使用时间和 pH 值等外部条件变化时,它可以不断地从产品中解离,污染水、食物、土壤、植物和沉积物等[2]。DEHP可经多种途径进入人体,如口鼻吸入、直接经口食入、皮肤吸收和在医疗活动中直接进入循环系统等[3]。DEHP具有疏水亲脂性,易从聚氯乙烯产品渗入至含脂肪类食物、含有脂质的溶液等,接触血液后更容易转移至血液[4]。因此,在接受塑料材料的侵入性医治(包括体外循环、体外膜肺氧合、透析和输血治疗)后,可以观察到患者体内DEHP水平升高。如接受输血治疗的成年人血液内DEHP浓度上升,为72.5~295.2 μg/mL(相当于185~755 μmol/L)[5]。 DEHP 及其代谢产物进入人体后会对内分泌系统、生殖系统、呼吸系统、神经系统等多个系统器官产生毒性作用[1]。多项流行病学调查研究表明DEHP 可以扩散到心脏,对心脏节律和心肌细胞产生影响,尚未有研究关注DEHP对心脏的滋养血管即心肌血管的影响[15-6]

  • 心肌血管是滋养心肌的血管,负责心肌中氧合血液和去氧血液的有效输送和排出[7]。所有心肌血管内衬一层单层、极性排列的内皮细胞,该层内皮细胞参与多种生理功能,如调节新血管形成、维持血管舒张性、维持血液流动性、调节血管壁通透性、抑制血管中促炎因子和凝血因子的激活等[8-9]。内皮细胞功能异常导致多种并发症,如凝血增强、细胞黏附、炎症激活、脂蛋白跨内皮运输及血管异常收缩和舒张等[10]。细胞凋亡是受到生物体严格调控的程序性死亡,能够维持各种组织和器官的稳定[11]。血管内皮细胞长期暴露在各种循环应激源的作用下,必须在凋亡和存活之间保持平衡,以保持内皮的完整性[12]。在病理条件下,内皮细胞凋亡是动脉粥样硬化的始动步骤,将导致随后的动脉粥样硬化发生发展[13]。已有研究表明DEHP暴露可以增加老年人群冠状动脉粥样硬化的风险[14]

  • 磷脂酰肌醇 3 ⁃ 激酶/蛋白激酶 B(phosphati⁃ dylinositol⁃3⁃kinase/protein kinase B,PI3K/AKT)信号通路存在于所有哺乳动物细胞中,并受细胞外信号的调控,对维持细胞正常功能起着至关重要的作用[15-16]。PI3K的激活可以磷酸化AKT,磷酸化的 AKT(p⁃AKT)可以进一步激活下游靶点调节细胞存活,包括Bcl⁃2家族蛋白(如Bcl⁃2、BAX)和半胱氨酸天冬氨酸蛋白酶家族蛋白(如Caspase⁃3)[17]。鉴于内皮细胞在血管中的重要地位,用不同剂量DEHP 处理人冠状动脉内皮细胞(human coronary artery endothelial cell,HCAEC),通过凋亡、血管形成等实验观察其对HCAEC功能的影响,并初步探索PI3K/ AKT信号通路在DEHP致HCAEC损伤中的作用。

  • 1 材料和方法

  • 1.1 材料

  • HCAEC(武汉 Fine Text 公司)。Dulbecco 改良 Eagle 培养基(Dulbecco’s Modified Eagle Medium, DMEM)、胎牛血清(fetal bovine serum,FBS)、青霉素和链霉素(Gibco公司,美国)。DEHP(Sigma⁃Aldrich 公司,美国)。CCK⁃8试剂和Annexin V⁃FITC/PI凋亡检测试剂盒(南京诺维赞公司)。抗 p⁃PI3、PI3K、 p⁃AKT、AKT抗体(Cell Signaling Technology公司,美国),抗 BAX、Bcl ⁃2、cleaved Caspase ⁃3、Caspase ⁃3、 GAPDH 抗体(上海艾比马特公司)。二甲基亚砜 (dimethyl sulfoxide,DMSO)(北京索莱宝公司)。磷酸酶抑制剂和蛋白酶抑制剂(Roche 公司,瑞士)。 Matrigel基质胶(Corning公司,美国)。

  • 1.2 方法

  • 1.2.1 细胞培养及DEHP染毒

  • 向高糖DMEM 中加入FBS和青霉素⁃链霉素双抗,使 FBS 和青霉素⁃链霉素双抗的终浓度分别为 10%和 1%,配制完全培养基。HCAEC 用完全培养基培养接种于无菌培养皿中,置于细胞培养箱常规培养,设定温度在37℃,CO2浓度在5%,每2 d更换 1次培养基。DEHP染毒实验,选取生长良好处于对数生长期的细胞,以DMSO为阴性对照组,实验组参考相关研究的染毒时间和 DEHP 浓度[518-20],将 DEHP 溶解稀释于 DMSO,再与培养基混合,将 DEHP 稀释成 128、256、384、512 μmol/L,使 HCAEC 染毒(DMSO终浓度低于0.1%),模拟DEHP暴露。

  • 1.2.2 细胞增殖⁃毒性实验

  • CCK⁃8试剂盒检测DEHP对细胞增殖能力的影响。将细胞以2 000个/孔的密度接种在96孔板中,过夜后用显微镜观察,细胞贴壁生长良好,弃去原有培养基后,设置空白对照组(无细胞仅有培养基)、阴性对照组(DMSO)、实验组(DEHP 128、256、 384、512 μmol/L),每组 5 个复孔,染毒后将 96 孔板放回培养箱继续培养12、24、48、72 h。染毒结束后,向每个孔加入10 μL CCK⁃8溶液,继续孵育2 h,2 h 后用酶标仪检测各组细胞在450 nm处吸光度值,各组结果表示为相应阴性对照组细胞活力的百分比,根据下列公式计算:(A-C)/(B-C)×100%(A:实验组吸光度值,B:阴性对照组吸光度值,C:空白对照组吸光度值)。

  • 1.2.3 体外成管实验

  • 制备细胞悬液,调整细胞密度为 1×106 个/mL。按照每孔50 μL将细胞悬液滴加到预先涂有基质胶的 96 孔板中,设置对照组(DMSO)、实验组(DEHP 128、256、384、512 μmol/L)。轻轻震荡,使细胞分布均匀。在培养箱中常规培养6 h。用倒置显微镜观察拍摄细胞成管情况,用 Image J 软件分析图片结果,定义至少3条管的交汇处为1个交点,以交点数目表示各组成管结果。

  • 1.2.4 流式细胞实验

  • 用 Annexin V⁃FITC/PI 细胞凋亡检测试剂盒检测DEHP对HCAEC凋亡的影响。6孔板常规培养细胞,设置对照组(DMSO)、实验组(DEHP 128、256、 384、512 μmol/L),在培养箱中常规培养染毒 72 h。染毒结束后,用不含乙二胺四乙酸(ethylene di⁃ amine tetraacetic acid,EDTA)的胰酶消化并收集细胞。细胞用预冷的磷酸盐缓冲液(phosphate buffer solution,PBS)漂洗 2 次后,用 100 μL 结合缓冲液重悬细胞,加入 5 μL Annexin V⁃FITC 和 5 μL PI 染色液室温(20~25℃)避光孵育10 min。孵育结束后,加入400 μL结合缓冲液,轻轻混匀。此外,用未经处理的细胞样品作为阴性对照,用DEHP 256 μmol/L组的细胞分别进行Annexin V⁃FITC和PI单染。

  • 在 1 h 内用流式细胞仪检测细胞样品,在双色流式细胞仪散点图上,早期凋亡细胞散在分布于右下象限,晚期凋亡细胞散在分布于右上象限。存活细胞和坏死细胞分别散布在左下象限和左上象限。定义各组结果为:凋亡率=早期凋亡率+晚期凋亡率。

  • 1.2.5 蛋白质印迹实验(Western blot)

  • 用 Western blot 评估凋亡相关蛋白(Bcl ⁃ 2、 cleaved Caspase ⁃3、Caspase ⁃3、BAX)及通路蛋白 (p⁃PI3K、PI3K、p⁃AKT、AKT)的表达水平。细胞染毒72 h后,吸去培养基,用预冷的PBS冲洗细胞。每组细胞用细胞裂解液(RIPA)裂解,裂解液中添加磷酸酶抑制剂和蛋白酶抑制剂。4℃裂解2 h后,将样品在 4℃下以 15 000 r/min 离心 20 min。使用增强的 BCA 蛋白质测定试剂盒测量上清液中蛋白质浓度,添加5×上样缓冲液后在100℃下变性15 min,分装冻存。通过 10%十二烷基硫酸钠聚丙烯酰胺凝胶电泳分离蛋白质,并转移至 0.45 μm 聚偏二氟乙烯(polyvinylidene fluoride,PVDF)膜。然后,5%牛血清白蛋白封闭缓冲液室温下封闭 2 h,按照蛋白 Marker 位置与目标蛋白分子量裁剪 PVDF 膜,与相应的一抗在 4℃下孵育过夜。抗体包括 Bcl⁃2 (1∶1 000)、BAX(1∶1 000)、cleaved Caspase⁃3 (1∶1 000)、Caspase⁃3(1∶1 000)、GAPDH(1∶ 1 000)、p⁃PI3K(1∶1 000)、PI3K(1∶1 000)、p⁃AKT (1∶1 000)、AKT(1∶1 000)。第2天在脱色摇床上用添加 0.1% Tween⁃20 的 Tris 缓冲盐水将 PVDF 膜洗涤 3 次,每次 15 min,在室温下二抗孵育 2 h,然后使用上述方法再次洗涤 3 次。使用增强的化学发光试剂,用自动化学发光图像分析系统曝光免疫反应条带。通过 Image J 软件 v1.53 测量每个条带的积分密度,分析相应样品中目的蛋白/GAPDH 或磷酸化蛋白/总蛋白的比例来计算相对蛋白表达含量。

  • 1.3 统计学方法

  • 用Excel 2019进行数据录入,SPSS 26.0进行数据的统计学分析,GraphPad Prism 8进行柱状图和折线图的绘制。本研究各组数据为正态分布且方差齐,结果以均数±标准差(x-±s)形式表示,两组间比较采用 t 检验,多组间比较采用单因素方差分析 (One⁃way ANOVA),组间两两比较采用 LSD 检验。以双侧α=0.05 为显著性检验标准,P <0.05 为差异有统计学意义。

  • 2 结果

  • 2.1 DEHP暴露对HCEAC细胞形态的影响

  • 倒置显微镜观察可见(图1A),72 h时,实验组和对照(DMSO)组细胞均为单层贴壁生长,呈现卵圆形或鹅卵石外观,边界清楚,胞浆丰富,细胞核清晰。实验组整体细胞密度较对照组减低,实验组小部分细胞胞浆内出现空泡状结构,细胞变大且结构松散,细胞核增大。

  • 2.2 DEHP抑制HCAEC的增殖

  • CCK⁃8 法观察 DEHP 对 HCAEC 增殖能力的影响。对DEHP浓度及染毒时间分别进行了单独效应检测。染毒12 h时,与对照(DMSO)组相比,实验组的细胞活力差异无统计学意义(P > 0.05)。染毒时间为24、48、72 h时,与对照(DMSO)组相比,实验组各浓度 DEHP(128、256、384、512 μmol/L)均能够抑制细胞的增殖能力(P < 0.05,图1B),随着DEHP浓度的增加,细胞活力依次降低,且 DEHP 128、256、 384、512 μmol/L组细胞活力相互之间的差异均有统计学意义(P < 0.05)。随着时间的延长,对照(DMSO)组细胞活力仅有轻微降低,各个时间点之间差异无统计学意义(P > 0.05,图1B)。随着染毒时间的延长,各实验组各时间点细胞活力依次降低,各时间点细胞活力相互之间的差异均有统计学意义(P < 0.05)。上述结果表明DEHP可抑制HCAEC 的增殖能力,并且染毒时间越长、DEHP浓度越高,毒性作用越明显,具有时间和浓度依赖性。

  • 2.3 DEHP抑制HCAEC血管形成能力

  • 体外成管实验能够在一定程度上模拟血管发生过程,通过观察细胞形成管腔的情况,评估DEHP 对 HCAEC 血管形成能力的影响。对照(DMSO)组细胞多呈现为拉长的梭形,互相连接成网状,形成的管状结构在 6 h 时趋于稳定,随着时间的延长逐渐崩解。实验组细胞多呈现孤立的圆形、卵圆形,散乱分布在视野中(图2A)。与对照(DMSO)组相比,DEHP 128 μmol/L组形成的交点数目减少,但是差异无统计学意义(P > 0.05)。DEHP 256、384、 512 μmol/L 组形成交点数目进一步减少,与对照 (DMSO)组相比,差异有统计学意义(P < 0.01,图2B)。以上结果说明 DEHP 可以抑制 HCAEC 的血管形成能力,具有浓度依赖性。

  • 图1 DEHP暴露对HCAEC细胞形态和增殖能力的影响

  • Figure1 Effects of DEHP exposure on cell morphology and proliferation of HCAEC

  • 细胞凋亡是由基因控制的细胞自主有序的死亡,为了评价DEHP 对HCAEC 凋亡的影响,进行了细胞流式实验。与对照(DMSO)组相比,各实验组凋亡细胞比例增加,差异具有统计学意义(P < 0.05,图3A、B)。后续检测了凋亡相关蛋白的表达水平, Western blot 实验结果显示,与对照组 Bcl⁃2 表达水平相比,各实验组Bcl⁃2表达水平降低,差异有统计学意义(P < 0.05,图3C、D);与对照(DMSO)组BAX 表达水平相比,DEHP 128 μmol/L 组 BAX 表达水平降低,但是其差异无统计学意义(P > 0.05)。DEHP 256、384、512 μmol/L组BAX表达水平增加,差异有统计学意义(P < 0.05);与对照(DMSO)组 cleaved Caspase⁃3/Caspase⁃3比值相比,各实验组比值上升,差异具有统计学意义(P < 0.05)。以上结果说明 DEHP 可以改变凋亡相关蛋白的表达水平,诱导HCAEC的凋亡。

  • 图2 DEHP暴露对HCAEC血管形成能力的影响

  • Figure2 The effect of DEHP exposure on the angiogenesis ability of HCAEC

  • 2.4 DEHP抑制PI3K/AKT信号通路

  • 为了探究上述结果的可能原因,进行了 West⁃ ern blot实验,各组PI3K、AKT水平差异无统计学意义(P > 0.05,图4A)。当 DEHP 浓度≥256 μmol/L 时,实验组p⁃PI3K/PI3K、p⁃AKT/AKT 比值较对照组降低,差异有统计学意义(P < 0.05,图4B)。上述结果说明,DEHP可以降低关键蛋白的磷酸化水平,抑制PI3K/AKT通路。

  • 3 讨论

  • 随着聚氯乙烯制品的广泛应用,DEHP 的产量逐年上升,年产量已超过200万吨[1]。DEHP是美国食品和药物协会批准的医疗器械中最常用的增塑剂,应用于储血袋、管道回路、肠内营养管、气管插管等。生活中进入人体的DEHP的量与生活方式和医疗器械的使用情况相关,接受医疗措施患者的血液 DEHP 浓度往往较高[21]。输血治疗后成年人血液 DEHP 浓度为 72.5~295.2 μg/mL,儿童输血治疗后血液 DEHP 浓度则更高,为 27.6~405 μg/mL(DEHP 100 μg/mL=256 μmol/L)[5]。成人冠状动脉搭桥和心脏移植手术DEHP暴露剂量分别为1.0、2.4 mg/(kg·d),成人和儿童进行体外循环手术时 DEHP的暴露量分别为3.0、14.0 mg/(kg·d),均超过人体耐受剂量即 0.6 mg/(kg·d)[22]。美国环保局数据显示,20 μg/(kg·d) 的DEHP暴露剂量即有造成肝脏肿大的风险,欧洲食品安全局报告称,50 μg/(kg·d)的DEHP暴露剂量可能具有睾丸毒性[1]

  • 近年,DEHP 的心血管毒性受到关注。研究显示,体外DEHP灌流大鼠心脏,可使其心率变慢、单位时间内冠脉流量下降[23]。流行病学调查显示, DEHP 暴露可导致青少年左室收缩压升高、心律失常[24-25],增加老年人群冠状动脉粥样硬化的风险,尿液 DEHP 水平亦与检测到的血管斑块回声成正相关[14]。对载脂蛋白 E 缺陷的小鼠给予 DEHP 灌胃,4周后小鼠表现出高脂血症、动脉粥样硬化加重和动脉内皮细胞的炎症损伤。动脉内皮细胞衬于血管内壁,它的凋亡被认为是动脉粥样硬化的始动因素之一,抑制内皮细胞凋亡可能是一种潜在的治疗动脉粥样硬化的策略[13]。因此,本研究根据临床患者可能的DEHP暴露情况和相关文献记载,建立了HCAEC的DEHP暴露模型[518-19],观察DEHP暴露对HCAEC的影响及可能机制。

  • 图3 DEHP暴露对HCAEC凋亡及凋亡相关蛋白表达情况的影响

  • Figure3 Effects of DEHP exposure on apoptosis and the expression of apoptosis⁃related proteins in HCAEC

  • 内皮细胞是心肌血管的重要组成部分,是血管快速新生和维持内皮层完整性的基础[26]。CCK⁃8结果表明,DEHP 能够以时间和剂量依赖的方式抑制 HCAEC 增殖能力。体外成管实验中,6 h时实验组交点数目比对照组减少,而染毒时间≤12 h 实验组和对照组增殖能力差异无统计学意义,说明DEHP 可以在不影响增殖的情况下对HCAEC血管形成能力产生抑制。

  • Western blot 结果显示实验组和对照组 PI3K、 AKT 差异无统计学意义。DEHP 浓度≥256 μmol/L 时,实验组p⁃PI3K/PI3K、p⁃AKT/AKT较对照组降低,差异有统计学意义,说明DEHP可降低PI3K/AKT信号通路关键蛋白的磷酸化。生理情况下PI3K的激活可以磷酸化和激活AKT,通过与其PH域结合,导致AKT移位到细胞膜的内表面[27-28]。磷酸化的AKT(p⁃AKT)可以进一步调节下游信号,如Bcl⁃2家族蛋白(如Bcl⁃2、BAX)和半胱氨酸天冬氨酸蛋白酶家族蛋白(如 Caspase⁃3)[29-30]。Caspase⁃3 在多条凋亡信号转导通路中发挥重要作用。Caspase⁃3以酶原形式存在于细胞质中,在细胞凋亡的早期被剪切激活,剪切的Caspase⁃3(cleaved Casepase⁃3)会引起一系列的级联反应,激活核酸内切酶,分解DNA片段,最终诱导细胞凋亡。Bcl⁃2和Bax是Bcl⁃2家族成员中的一对凋亡调控基因,参与细胞线粒体凋亡途径的调控机制。Bax 和 Bcl⁃2 主要作用于线粒体膜。 Bax 是一种促凋亡基因,可促进线粒体释放细胞色素 C,从而促进细胞凋亡。Bcl⁃2 是一种抗凋亡基因,可以抑制细胞色素C的释放,并拮抗Bax的促凋亡作用[31-32]。本研究结果显示,与对照(DMSO)组相比,实验组Bcl⁃2降低,同时BAX和cleaved Caspase⁃ 3/Caspase⁃3增加,与对照组差异有统计学意义(P < 0.05)。此结果与流式细胞实验相符,与对照 (DMSO)组相比,实验组细胞凋亡比例上升,差异有统计学意义(P <0.05)。DEHP对HCAEC凋亡的诱导作用,恰恰反映其毒性的可调控性,从细胞凋亡角度,PI3K/AKT参与DEHP诱导的HCAEC凋亡,提示其可能作为将来治疗研究的一个切入点。

  • 图4 DEHP对PI3K/AKT信号通路的影响

  • Figure4 Effects of DEHP on PI3K/AKT signaling pathway

  • 本研究表明,以临床患者可能接触的DEHP剂量作用于 HCEAC,可以通过抑制 PI3K/AKT 信号通路诱导其凋亡,抑制其增殖、成管能力(图5),这将为寻求临床 DEHP 暴露致 HCAEC 损伤的治疗方法提供理论基础。

  • 图5 DEHP通过抑制PI3K/AKT信号通路抑制HCAEC增殖、成管能力,诱导其凋亡

  • Figure5 DEHP inhibits the proliferation and tube forma⁃ tion,induces apoptosis of HCAEC by inhibiting PI3K/AKT signaling pathway

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  • 参考文献

    • [1] ROWDHWAL S S S,CHEN J.Toxic effects of Di⁃2⁃ethyl⁃ hexyl phthalate:an overview[J].Biomed Res Int,2018,2018:1750368

    • [2] ARUKWE A,IBOR O R,ADEOGUN A O.Biphasic mod⁃ ulation of neuro ⁃ and interrenal steroidogenesis in juve⁃ nile African sharptooth catfish(Clarias gariepinus)ex⁃ posed to waterborne di ⁃(2⁃ethylhexyl)phthalate[J].Gen Comp Endocrinol,2017,254:22-37

    • [3] AMARA I,TIMOUMI R,ANNABI E,et al.Di(2⁃ ethyl⁃ hexyl)phthalate induces cardiac disorders in BALB/c mice[J].Environ Sci Pollut Res Int,2019,26(8):7540-7549

    • [4] INOUE K,HIGUCHI T,OKADA F,et al.The validation of column ⁃ switching LC/MS as a high ⁃ throughput ap⁃ proach for direct analysis of di(2⁃ethylhexyl)phthalate re⁃ leased from PVC medical devices in intravenous solution [J].J Pharm Biomed Anal,2003,31(6):1145-1152

    • [5] JAIMES R,MCCULLOUGH D,SIEGEL B,et al.Plasticiz⁃ er interaction with the heart:chemicals used in plastic medical devices can interfere with cardiac electrophysiol⁃ ogy[J].Circ Arrhythm Electrophysiol,2019,12(7):e007294

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