Division of Biochemical Toxicology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR, 72079, USA.
Division of Bioinformatics and Biostatistics, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR, 72079, USA.
Arch Toxicol. 2017 Jun;91(6):2405-2423. doi: 10.1007/s00204-016-1893-6. Epub 2016 Nov 28.
Triclosan is a widely used broad-spectrum anti-bacterial agent. The objectives of this study were to identify which cytochrome P450 (CYP) isoforms metabolize triclosan and to examine the effects of CYP-mediated metabolism on triclosan-induced cytotoxicity. A panel of HepG2-derived cell lines was established, each of which overexpressed a single CYP isoform, including CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP3A7, CYP4A11, and CYP4B1. The extent of triclosan metabolism by each CYP was assessed by reversed-phase high-performance liquid chromatography with online radiochemical detection. Seven isoforms were capable of metabolizing triclosan, with the order of activity being CYP1A2 > CYP2B6 > CYP2C19 > CYP2D6 ≈ CYP1B1 > CYP2C18 ≈ CYP1A1. The remaining 11 isoforms (CYP2A6, CYP2A7, CYP2A13, CYP2C8, CYP2C9, CYP2E1, CYP3A4, CYP3A5, CYP3A7, CYP4A11, and CYP4B1) had little or no activity toward triclosan. Three metabolites were detected: 2,4-dichlorophenol, 4-chlorocatechol, and 5'-hydroxytriclosan. Consistent with the in vitro screening data, triclosan was extensively metabolized in HepG2 cells overexpressing CYP1A2, CYP2B6, CYP2C19, CYP2D6, and CYP2C18, and these cells were much more resistant to triclosan-induced cytotoxicity compared to vector cells, suggesting that CYP-mediated metabolism of triclosan attenuated its cytotoxicity. In addition, 2,4-dichlorophenol and 4-chlorocatechol were less toxic than triclosan to HepG2/vector cells. Conjugation of triclosan, catalyzed by human glucuronosyltransferases (UGTs) and sulfotransferases (SULTs), also occurred in HepG2/CYP-overexpressing cells and primary human hepatocytes, with a greater extent of conjugation being associated with higher cell viability. Co-administration of triclosan with UGT or SULT inhibitors led to greater cytotoxicity in HepG2 cells and primary human hepatocytes, indicating that glucuronidation and sulfonation of triclosan are detoxification pathways. Among the 18 CYP-overexpressing cell lines, an inverse correlation was observed between cell viability and the level of triclosan in the culture medium. In conclusion, human CYP isoforms that metabolize triclosan were identified, and the metabolism of triclosan by CYPs, UGTs, and SULTs decreased its cytotoxicity in hepatic cells.
三氯生是一种广泛使用的广谱抗菌剂。本研究的目的是确定哪些细胞色素 P450（CYP）同工酶代谢三氯生，并研究 CYP 介导的代谢对三氯生诱导的细胞毒性的影响。建立了一组 HepG2 衍生的细胞系，每个细胞系都过表达一种单一的 CYP 同工酶，包括 CYP1A1、CYP1A2、CYP1B1、CYP2A6、CYP2A7、CYP2A13、CYP2B6、CYP2C8、CYP2C9、CYP2C18、CYP2C19、CYP2D6、CYP2E1、CYP3A4、CYP3A5、CYP3A7、CYP4A11 和 CYP4B1。通过反相高效液相色谱法与在线放射性化学检测评估每种 CYP 代谢三氯生的程度。七种同工酶能够代谢三氯生，活性顺序为 CYP1A2>CYP2B6>CYP2C19>CYP2D6≈CYP1B1>CYP2C18≈CYP1A1。其余 11 种同工酶（CYP2A6、CYP2A7、CYP2A13、CYP2C8、CYP2C9、CYP2E1、CYP3A4、CYP3A5、CYP3A7、CYP4A11 和 CYP4B1）对三氯生几乎没有或没有活性。检测到三种代谢物：2,4-二氯苯酚、4-氯邻苯二酚和 5'-羟基三氯生。与体外筛选数据一致，CYP1A2、CYP2B6、CYP2C19、CYP2D6 和 CYP2C18 过表达的 HepG2 细胞中三氯生被广泛代谢，与载体细胞相比，这些细胞对三氯生诱导的细胞毒性的抵抗力更强，表明 CYP 介导的三氯生代谢减弱了其细胞毒性。此外，2,4-二氯苯酚和 4-氯邻苯二酚对 HepG2/载体细胞的毒性小于三氯生。三氯生的葡萄糖醛酸转移酶（UGTs）和磺基转移酶（SULTs）催化的共轭也发生在 HepG2/CYP 过表达细胞和原代人肝细胞中，与更高的细胞活力相关的共轭程度更高。三氯生与 UGT 或 SULT 抑制剂共同给药导致 HepG2 细胞和原代人肝细胞的细胞毒性增加，表明三氯生的葡萄糖醛酸化和磺化是解毒途径。在 18 种 CYP 过表达细胞系中，观察到细胞活力与培养基中三氯生浓度之间存在反比关系。总之，鉴定了代谢三氯生的人细胞色素 P450 同工酶，CYP、UGT 和 SULT 对三氯生的代谢降低了其在肝细胞中的细胞毒性。
