Abstract：
Currently, there is no test system, whether in vitro or in vivo, capable of examining all endpoints required for genotoxicity evaluation used in pre-clinical drug safety assessment. The objective of this study was to develop a model which could assess all the required endpoints and possesses robust human metabolic activity, that could be used in a streamlined, animal-free manner. Liver-on-chip (LOC) models have intrinsic human metabolic activity that mimics the in vivo environment, making it a preferred test system. For our assay, the LOC was assembled using primary human hepatocytes or HepaRG cells, in a MPS-T12 plate, maintained under microfluidic flow conditions using the PhysioMimix® Microphysiological System (MPS), and co-cultured with human lymphoblastoid (TK6) cells in transwells. This system allows for interaction between two compartments and for the analysis of three different genotoxic endpoints, i.e. DNA strand breaks (comet assay) in hepatocytes, chromosome loss or damage (micronucleus assay) and mutation (Duplex Sequencing) in TK6 cells. Both compartments were treated at 0, 24 and 45 h with two direct genotoxicants: methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS), and two genotoxicants requiring metabolic activation: benzo[a]pyrene (B[a]P) and cyclophosphamide (CP). Assessment of cytochrome activity, RNA expression, albumin, urea and lactate dehydrogenase production, demonstrated functional metabolic capacities. Genotoxicity responses were observed for all endpoints with MMS and EMS. Increases in the micronucleus and mutations (MF) frequencies were also observed with CP, and %Tail DNA with B[a]P, indicating the metabolic competency of the test system. CP did not exhibit an increase in the %Tail DNA, which is in line with in vivo data. However, B[a]P did not exhibit an increase in the % micronucleus and MF, which might require an optimization of the test system. In conclusion, this proof-of-principle experiment suggests that LOC-MPS technology is a promising tool for in vitro hazard identification genotoxicants.
摘要：
目前,没有测试系统,无论是体外还是体内，能够检查临床前药物安全性评估中使用的遗传毒性评估所需的所有终点。这项研究的目的是开发一个模型，可以评估所有所需的终点，并具有强大的人体代谢活动，可以用在流线型的，无动物的方式。肝脏芯片（LOC）模型具有内在的人类代谢活动，模拟体内环境，使其成为首选的测试系统。对于我们的检测，LOC是使用原代人肝细胞或HepaRG细胞组装的，在MPS-T12板中，使用PhysioMimix®微生理系统(MPS)在微流体流动条件下保持，并在transwell中与人淋巴母细胞（TK6）细胞共培养。该系统允许两个区室之间的相互作用，并分析三个不同的基因毒性终点，即肝细胞中的DNA链断裂（彗星测定），TK6细胞的染色体丢失或损伤（微核分析）和突变（双重测序）。两个隔室分别在0、24和45小时用两种直接遗传毒性剂处理：甲磺酸甲酯（MMS）和甲磺酸乙酯（EMS），和两种需要代谢激活的基因毒性：苯并[a]芘（B[a]P）和环磷酰胺（CP）。细胞色素活性的评估，RNA表达，白蛋白,尿素和乳酸脱氢酶的生产，表现出功能性代谢能力。用MMS和EMS观察到所有终点的遗传毒性反应。CP也观察到微核和突变（MF）频率的增加，和%带有B[a]P的尾部DNA，指示测试系统的代谢能力。CP没有表现出%尾DNA的增加，这与体内数据一致。然而,B[a]P的微核和MF百分比没有增加，这可能需要优化测试系统。总之,这项原理验证实验表明,LOC-MPS技术是一种有前景的体外危险识别基因毒性剂的工具.
