Biochemistry Department, University College Cork, College Road, Cork, Ireland.
Environ Sci Technol. 2010 Apr 1;44(7):2535-41. doi: 10.1021/es903157h.
Microcystins (MC) are potent hepatic toxins delivered into the cells by organic anion transporting peptides (OATP) where they target protein phosphatases and mitochondria. We analyzed the effects of MC-LR on primary hepatocytes, HepG2, and Jurkat T cells, and isolated rat liver mitochondria by measuring changes in O(2) consumption by optical oxygen sensing technique. Respiration of fresh primary hepatocytes was inhibited by MC-LR with EC50 = 2.74 +/- 0.65 nM, whereas an uncoupling effect on mitochondrial state 2 and state 3 respiration was observed with glutamate/malate as a substrate. HepG2 and Jurkat T cells lacking OATP showed no sensitivity to MC-LR; however, facilitated delivery of MC-LR resulted in a marked enhancement of HepG2 O(2) consumption and inhibition of Jurkat O(2) consumption at >or=0.1 nM. The respiratory response did not coincide with changes in viability, total cellular ATP, extracellular acidification, ROS formation, or protein phosphorylation, which were detectable at higher MC-LR doses. Such prominent effect on cellular respiration was therefore used for the detection of MC-LR in environmental samples. A simple and sensitive screening assay for MC-LR toxicity was developed, which uses Jurkat cells, facilitated delivery of the toxin(s) and measurement on a fluorescent reader. The assay was applied to a panel of environmental samples suspected to contain MC and benchmarked against the ELISA test. It allowed identification of toxic samples and quantification of both nonspecific and MC-LR type of toxicity.
微囊藻毒素（MC）是通过有机阴离子转运多肽（OATP）进入细胞的强效肝毒素，在细胞内，它们靶向蛋白磷酸酶和线粒体。我们通过光学氧感应技术测量 O2 消耗的变化，分析了 MC-LR 对原代肝细胞、HepG2 和 Jurkat T 细胞的影响，并分离了大鼠肝线粒体。MC-LR 对新鲜原代肝细胞的呼吸作用的 EC50 值为 2.74 +/- 0.65 nM，而谷氨酸/苹果酸作为底物时观察到线粒体状态 2 和状态 3 呼吸的解偶联作用。缺乏 OATP 的 HepG2 和 Jurkat T 细胞对 MC-LR 没有敏感性；然而，MC-LR 的易位作用导致 HepG2 的 O2 消耗明显增强，而 Jurkat 的 O2 消耗则在 >or=0.1 nM 时受到抑制。呼吸反应与细胞活力、总细胞 ATP、细胞外酸化、ROS 形成或蛋白质磷酸化的变化不一致，这些变化在更高剂量的 MC-LR 下才能检测到。因此，这种对细胞呼吸的显著影响被用于检测环境样本中的 MC-LR。开发了一种简单而灵敏的 MC-LR 毒性筛选检测方法，该方法使用 Jurkat 细胞、毒素的易位作用和荧光读取器进行测量。该检测方法应用于一组疑似含有 MC 的环境样本，并与 ELISA 测试进行了基准测试。它能够识别有毒样本，并对非特异性和 MC-LR 型毒性进行定量。
