Metabolic activation of hepatotoxic drug (benzbromarone) induced mitochondrial membrane permeability transition.
Metabolic activation of hepatotoxic drug (benzbromarone) induced mitochondrial membrane permeability transition.
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肝毒性药物（苯溴马隆）的代谢激活诱导线粒体膜通透性转变。
DOI：
10.1016/j.taap.2015.06.018
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Ito K.
中科院分区：
文献类型：
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作者：
Shirakawa M;Sekine S;Tanaka A;Horie T;Ito K.
药物性肝损伤（DILI）的风险是制药行业非常关注的问题。众所周知，药物代谢激活形成毒性代谢物（TMs）与DILI发病密切相关。药物诱导的线粒体功能障碍也与DILI风险增加密切相关。然而，由于TMs的鉴定和纯化困难，很难确定与DILI恶化相关的TMs的靶点。在这项研究中，我们提出了一个连续的体外检测系统来评估TM的形成及其在一锅过程中诱导线粒体通透性转变（MPT）的能力。在这个实验系统中，新鲜分离的大鼠肝脏线粒体与44种试验药物的反应溶液一起在存在或不存在NADPH的情况下与肝微粒体预孵育；然后，nadph依赖的MPT孔开放被评估为线粒体肿胀。在这个实验系统中，几种肝毒性药物，包括苯溴马隆（BBR），以nadph依赖的方式显著诱导MPT。我们研究了使用BBR作为模型药物的合理性，因为它在我们的分析系统中显示出最突出的MPT。几种细胞色素P450 （CYP）抑制剂（克霉唑和SKF-525A， 100 μM）均可抑制BBR候选毒性代谢物（1′，6-(OH)2BBR）和nadph依赖性MPT的产生。总之，该检测系统可用于评估综合代谢物依赖的MPT，而无需鉴定或纯化代谢物。
The risk of drug-induced liver injury (DILI) is of great concern to the pharmaceutical industry. It is well-known that metabolic activation of drugs to form toxic metabolites (TMs) is strongly associated with DILI onset. Drug-induced mitochondrial dysfunction is also strongly associated with increased risk of DILI. However, it is difficult to determine the target of TMs associated with exacerbation of DILI because of difficulties in identifying and purifying TMs. In this study, we propose a sequential in vitro assay system to assess TM formation and their ability to induce mitochondrial permeability transition (MPT) in a one-pot process. In this assay system, freshly-isolated rat liver mitochondria were incubated with reaction solutions of 44 test drugs preincubated with liver microsomes in the presence or absence of NADPH; then, NADPH-dependent MPT pore opening was assessed as mitochondrial swelling. In this assay system, several hepatotoxic drugs, including benzbromarone (BBR), significantly induced MPT in a NADPH-dependent manner. We investigated the rationality of using BBR as a model drug, since it showed the most prominent MPT in our assay system. Both the production of a candidate toxic metabolite of BBR (1′,6-(OH)2BBR) and NADPH-dependent MPT were inhibited by several cytochrome P450 (CYP) inhibitors (clotrimazole and SKF-525A, 100 μM). In summary, this assay system can be used to evaluate comprehensive metabolite-dependent MPT without identification or purification of metabolites.