Di-2-ethylhexyl phthalate (DEHP) is frequently used as a plasticizer for wrapping films, in toys, and in medical devices. Previous studies demonstrated that DEHP in mouse reduced testicular and epididymis weights, suppressed levels of serum testosterone, luteinizing hormone, and follicle-stimulating hormone, and decreased synthesis of testosterone by Leydig cells. Due to these anti-androgenic effects of DEHP on the reproductive system, the aim of this study was to examine whether substitutes such as acetyl triethyl citrate (ATEC) and acetyl tributyl citrate (ATBC) also damaged the reproductive system. In particular, this study investigated the anti-androgenic effects and cytotoxicity of DEHP substitutes using castrated male Sprague--Dawley rats employing the in vivo Hershberger assay and in vitro mouse Leydig (TM3) cells and mouse fibroblast (NIH-3T3) cell lines. In the Hershberger assay, rats were administered testosterone propionate and ATEC or ATBC at 20, 100, or 500 mg/kg b.w./day or DEHP (500 mg/kg b.w./day). Controls received testosterone antagonist flutamide (positive control), testosterone only (negative control), or corn oil only (vehicle control). ATEC/ATBC treatment produced no significant differences compared with testosterone in 5-androgen-dependent tissues weights including ventral prostate, seminal vesicles, levator ani-bulbocavernosus muscle, Cowper’s glands, and glans penis. In the 500 mg/kg ATBC group, there was a significant reduction in liver weight. The MTT assay revealed that cell viability of both TM3 and NIH-3T3 cells treated with ATEC was not markedly altered. However, ATBC significantly reduced TM3 and NIH-3T3 cell viability in a concentration-dependent manner. Further, ATBC reduced cell viability to greater extent in TM3 versus NIH-3T3 cells. Based upon the observed effects of citrate ester substitutes on reproductive tissue responses and cytotoxicity, ATEC compared to ATBC may be a better alternative to DEHP for potential commercial uses.

Abbreviations

ATEC: acetyl triethyl citrate; ATBC: acetyl tributyl citrate; CG: Cowper’s glands; DEHP: di-2-ethylhexyl phthalate; DMEM: Dulbecco’s modified Eagle’s medium; DMSO: dimethyl sulfoxide; GP: glans penis; LABC: levator ani-bulbocavernosus muscle; MTT: methyl tetrazolium; NC: negative control; NT: untreated control; PC: positive control; SV: seminal vesicle; TP: testosterone propionate; VC: vehicle control; VP: ventral prostate.

中文翻译：

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柠檬酸酯替代邻苯二甲酸二-2-乙基己酯：体内生殖和体外细胞毒性评估。

邻苯二甲酸二-2-乙基己酯（DEHP）经常用作包装薄膜，玩具和医疗设备中的增塑剂。先前的研究表明，小鼠中的DEHP降低了睾丸和附睾的重量，抑制了血清睾丸激素，促黄体生成激素和促卵泡激素的水平，并减少了Leydig细胞合成睾丸激素的能力。由于DEHP对生殖系统具有这些抗雄激素作用，因此本研究的目的是检查诸如柠檬酸乙酰三乙酯（ATEC）和柠檬酸乙酰三丁酯（ATBC）之类的替代品是否也损害了生殖系统。尤其是，本研究使用体内Hershberger分析和体外方法，使用去势雄性Sprague-Dawley大鼠研究了DEHP替代品的抗雄激素作用和细胞毒性。小鼠Leydig（TM3）细胞和小鼠成纤维细胞（NIH-3T3）细胞系。在Hershberger分析中，以20、100或500 mg / kg bw /天或DEHP（500 mg / kg bw /天）的剂量给大鼠丙酸睾丸酮和ATEC或ATBC。对照组接受睾丸激素拮抗剂氟他胺（阳性对照），仅睾丸激素（阴性对照）或仅玉米油（载体对照）。与睾丸激素相比，ATEC / ATBC治疗与5雄激素依赖性组织的重量（包括腹侧前列腺，精囊，提肛肌，膀胱角肌，考珀氏腺和龟头阴茎）的重量没有显着差异。在500 mg / kg ATBC组中，肝脏重量显着减少。MTT分析显示，经ATEC处理的TM3和NIH-3T3细胞的细胞活力均未显着改变。然而，ATBC以浓度依赖性方式显着降低TM3和NIH-3T3细胞的活力。此外，与NIH-3T3细胞相比，ATBC在TM3中更大程度地降低了细胞活力。根据观察到的柠檬酸酯替代物对生殖组织反应和细胞毒性的影响，与ATBC相比，ATEC可能是DEHP更好的替代品，具有潜在的商业用途。

缩略语

ATEC：乙酰柠檬酸三乙酯；ATBC：乙酰柠檬酸三丁酯；CG：考珀氏腺；DEHP：邻苯二甲酸二-2-乙基己酯；DMEM：Dulbecco改良的Eagle培养基；DMSO：二甲基亚砜；GP：龟头阴茎；LABC：提肛苯丁酸肌；MTT：甲基四唑鎓；NC：阴性对照；NT：未经处理的对照；PC：阳性对照；SV：精囊；TP：丙酸睾丸酮；VC：车辆控制；VP：腹侧前列腺。