Zhao F, Mayura K, Hutchinson R W, Lewis R P, Burghardt R C, Phillips T D
Department of Veterinary Anatomy and Public Health, College of Veterinary Medicine, Texas A&M University, College Station 77843-4468, USA.
Toxicol Lett. 1995 Jun;78(1):35-42. doi: 10.1016/0378-4274(95)99684-a.
The chlorophenols (CPs) comprise a major class of widely distributed and frequently occurring environmental contaminants. Previous studies have demonstrated the adverse effects of CPs on embryonic and fetal development. HEPM (human embryonic palatal mesenchymal) and MOT (mouse ovarian tumor) cell lines have been utilized in complementary bioassays for the detection of teratogens, but not the CPs. In this study, our objectives were 2-fold: (1) to determine if the HEPM assay could be used to complement other bioassay systems of nonhuman origin, i.e., Hydra attenuata (HA) and rat whole embryo culture (WEC), in the evaluation of the developmental toxicity of CPs, and (2) to delineate the ability of the HEPM assay to evaluate structure-activity relationships of pentachlorophenol (C5P), 2,3,4,5-tetrachlorophenol (C4P), 2,3,5-trichlorophenol (C3P), 3,5-dichlorophenol (C2P), 4-monochlorophenol (CP), phenol, and CP derivatives (i.e., acetates, sodium phenates and anisoles). HEPM cells were seeded into each well of a 24-well plate and cultivated for 24 h. The medium was replaced with fresh medium containing various concentrations of test chemicals dissolved in dimethyl sulfoxide (DMSO, 0.1%). After culturing for 72 h, the medium was removed, cells were trypsinized, and cell number determined. The HEPM cell growth inhibition assay demonstrated a linear relationship between the IC50 values of the CPs and degree of chlorine substitution. The IC50 values of C5P, C4P, C3P, C2P, CP, and phenol were 18.8, 21.5, 27.5, 63.0, 150.0 and 470.0 microM, respectively. A clear structure-activity relationship was observed between toxicity of CPs and the degree of chlorine substitution. The rank order of CP toxicity from the HEPM assay (i.e., C5P > C4P > C3P > C2P > CP > phenol) is in excellent agreement with previous in vitro and in vivo studies. However, contrary to published reports, the HEPM assay predicted that all CPs were teratogenic (false positives). These findings suggest that the HEPM cell growth inhibition bioassay may be useful to discriminate between subtle differences in structure-activity and, in combination with other bioassays, might facilitate the rapid detection and prioritization of diverse cytotoxins, including various developmental toxicants. Importantly, conclusions about the teratogenicity of a test chemical (via HEPM testing) should be approached with caution and confirmed with other teratogen-sensitive systems.
氯酚（CPs）是一类分布广泛且频繁出现的主要环境污染物。先前的研究已证明氯酚对胚胎和胎儿发育具有不良影响。人胚胎腭间充质（HEPM）和小鼠卵巢肿瘤（MOT）细胞系已用于检测致畸剂的补充生物测定，但未用于氯酚的检测。在本研究中，我们的目标有两个：（1）确定HEPM测定法是否可用于补充其他非人类来源的生物测定系统，即水螅（HA）和大鼠全胚胎培养（WEC），以评估氯酚的发育毒性；（2）描绘HEPM测定法评估五氯酚（C5P）、2,3,4,5 - 四氯酚（C4P）、2,3,5 - 三氯酚（C3P）、3,5 - 二氯酚（C2P）、4 - 一氯酚（CP）、苯酚和CP衍生物（即乙酸酯、苯酚钠和苯甲醚）的构效关系的能力。将HEPM细胞接种到24孔板的每个孔中并培养24小时。用含有溶解于二甲基亚砜（DMSO，0.1%）中的各种浓度测试化学品的新鲜培养基替换培养基。培养72小时后，去除培养基，将细胞用胰蛋白酶消化，并测定细胞数量。HEPM细胞生长抑制测定表明氯酚的半数抑制浓度（IC50）值与氯取代程度之间存在线性关系。C5P、C4P、C3P、C2P、CP和苯酚的IC50值分别为18.8、21.5、27.5、63.0、150.0和470.0微摩尔。在氯酚的毒性与氯取代程度之间观察到明显的构效关系。HEPM测定法得出的氯酚毒性排序（即C5P > C4P > C3P > C2P > CP > 苯酚）与先前的体外和体内研究结果非常一致。然而，与已发表的报告相反，HEPM测定法预测所有氯酚均具有致畸性（假阳性）。这些发现表明，HEPM细胞生长抑制生物测定法可能有助于区分构效关系中的细微差异，并且与其他生物测定法结合使用，可能有助于快速检测和优先排序各种细胞毒素，包括各种发育毒物。重要的是，关于测试化学品致畸性的结论（通过HEPM测试）应谨慎得出，并用其他致畸剂敏感系统进行确认。
