Broekman Mark M T J, Roelofs Hennie M J, Wong Dennis R, Kerstholt Mariska, Leijten Alex, Hoentjen Frank, Peters Wilbert H M, Wanten Geert J A, de Jong Dirk J
Department of Gastroenterology, Radboud University Nijmegen Medical Center, 455, P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands,
Cell Biol Toxicol. 2015 Jun;31(3):161-71. doi: 10.1007/s10565-015-9301-1. Epub 2015 Apr 28.
The use of thiopurines is frequently accompanied by hepatotoxicity. Studies on hepatocyte cultures showed a time- and dose-dependent increase of thiopurine toxicity. 5-Aminosalicylic acid (5-ASA) and allopurinol can influence thiopurine metabolism; however, it is unknown whether this affects in vitro cytotoxicity.
Human hepatoma cells (Huh7, HepG2 and HepaRG) were incubated with increasing concentrations of thiopurines, 5-ASA or allopurinol. Water-soluble tetrazolium salt-1 (WST-1) cytotoxicity assays were used to calculate cell survival curves and half maximal inhibitory concentrations (IC50). Combination experiments with thiopurines with a fixed dose of 200 μM 5-ASA or 100 μM allopurinol were conducted in HepaRG cells. Caspase-3/7 activation was evaluated, and single cell electrophoresis analysis was performed.
A time- and dose-related cytotoxic effect was seen with azathioprine (AZA) in all hepatoma cells, whereas Huh7 and HepG2 cells did not show toxicity to 6-mercaptopurine (6-MP). HepaRG cells expressed the highest levels of drug metabolising enzymes, and therefore, combination experiments were conducted in HepaRG cells. Addition of a non-toxic dose of allopurinol resulted in a twofold to threefold increased cytotoxicity of all thiopurines, which seemed to be mediated by apoptosis/DNA damage.
The addition of allopurinol to thiopurines leads to a two-threefold increased cytotoxicity in HepaRG cells.
硫唑嘌呤的使用常伴有肝毒性。肝细胞培养研究显示硫唑嘌呤毒性呈时间和剂量依赖性增加。5-氨基水杨酸（5-ASA）和别嘌醇可影响硫唑嘌呤代谢；然而，这是否会影响体外细胞毒性尚不清楚。
将人肝癌细胞（Huh7、HepG2和HepaRG）与浓度不断增加的硫唑嘌呤、5-ASA或别嘌醇一起孵育。采用水溶性四氮唑盐-1（WST-1）细胞毒性试验计算细胞存活曲线和半数最大抑制浓度（IC50）。在HepaRG细胞中进行硫唑嘌呤与固定剂量200μM 5-ASA或100μM别嘌醇的联合实验。评估半胱天冬酶-3/7的激活情况，并进行单细胞电泳分析。
在所有肝癌细胞中均观察到硫唑嘌呤（AZA）具有时间和剂量相关的细胞毒性作用，而Huh7和HepG2细胞对6-巯基嘌呤（6-MP）无毒性。HepaRG细胞表达的药物代谢酶水平最高，因此在HepaRG细胞中进行了联合实验。添加无毒剂量的别嘌醇会使所有硫唑嘌呤的细胞毒性增加两倍至三倍，这似乎是由细胞凋亡/DNA损伤介导的。
在硫唑嘌呤中添加别嘌醇会使HepaRG细胞的细胞毒性增加两到三倍。
