Sato Brittany L, Ward Monika A, Astern Joshua M, Kendal-Wright Claire E, Collier Abby C
Cellular and Molecular Biology, John A. Burns School of Medicine, University of Hawaii, 651 Ilalo Street, Honolulu, HI 96813, USA; Tropical Medicine, Medical Microbiology and Pharmacology, John A. Burns School of Medicine, University of Hawaii, 651 Ilalo Street, Honolulu, HI 96813, USA; Natural Sciences and Mathematics, Chaminade University of Honolulu, 3140 Waialae Avenue, Honolulu, HI 96816, USA.
Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii, 1960 East-West Road, Honolulu, HI 96822, USA.
Toxicol In Vitro. 2015 Feb;29(1):103-12. doi: 10.1016/j.tiv.2014.09.008. Epub 2014 Oct 2.
Human primary placental explant culture is well established for cytokine signaling and toxicity, but has not been validated for steroidogenic or metabolic toxicology. The technique has never been investigated in the mouse. We characterized human and mouse placental explants for up to 96 h in culture. Explant viability (Lactate dehydrogenase) and sex steroid levels were measured in media using spectrophotometry and ELISA, respectively. Expression and activities of the steroidogenic (3β-hydroxysteroid dehydrogenase, Cytochrome P45017A1, Cytochrome P45019), conjugation (UDP-glucuronosyltransferase, sulfotransferase (SULT)), and regeneration (β-glucuronidase, arylsulfatase C (ASC)) enzymes were determined biochemically in tissues with fluorimetric and spectrophotometric assays, and western blot. Explants were viable up to 96 h, but progesterone, estrone, and 17β-estradiol secretion decreased. Steroidogenic enzyme expression and activities were stable in mouse explants and similar to levels in freshly isolated tissues, but were lower in human explants than in fresh tissue (P<0.01). Human and mouse explants exhibited significantly less conjugation after 96 h, SULT was not detected in the mouse, and neither explants had active ASC, although proteins were expressed. Mouse explants may be useful for steroid biochemistry and endocrine disruption studies, but not metabolic conjugation. In contrast, human explants may be useful for studying conjugation for <48 h, but not for steroid/endocrine studies.
人胎盘原代外植体培养在细胞因子信号传导和毒性研究方面已得到充分确立，但尚未在类固醇生成或代谢毒理学方面得到验证。该技术从未在小鼠中进行过研究。我们对人胎盘和小鼠胎盘外植体进行了长达96小时的培养特性研究。分别使用分光光度法和酶联免疫吸附测定法测量培养基中外植体的活力（乳酸脱氢酶）和性类固醇水平。通过荧光和分光光度测定法以及蛋白质印迹法对组织中的类固醇生成酶（3β - 羟基类固醇脱氢酶、细胞色素P45017A1、细胞色素P45019）、结合酶（尿苷二磷酸葡萄糖醛酸基转移酶、磺基转移酶（SULT））和再生酶（β - 葡萄糖醛酸酶、芳基硫酸酯酶C（ASC））的表达和活性进行生化测定。外植体在长达96小时内保持活力，但孕酮、雌酮和17β - 雌二醇的分泌减少。类固醇生成酶的表达和活性在小鼠外植体中保持稳定，与新鲜分离组织中的水平相似，但在人外植体中低于新鲜组织（P<0.01）。人胎盘和小鼠胎盘外植体在培养96小时后结合能力均显著降低，在小鼠中未检测到SULT，并且两种外植体均没有活性ASC，尽管有蛋白质表达。小鼠外植体可能有助于类固醇生物化学和内分泌干扰研究，但不适用于代谢结合研究。相比之下，人外植体可能有助于研究<48小时的结合情况，但不适用于类固醇/内分泌研究。
