PDF(Pubmed)
Abstract：
Amanita phalloides poisonings account for the majority of fatal mushroom poisonings. Recently, we identified hematotoxicity as a relevant aspect of Amanita poisonings. In this study, we investigated the effects of the main toxins of Amanita phalloides, α- and β-amanitin, on hematopoietic cell viability in vitro. Hematopoietic cell lines were exposed to α-amanitin or β-amanitin for up to 72 h with or without the pan-caspase inhibitor Z-VAD(OH)-FMK, antidotes N-acetylcysteine, silibinin, and benzylpenicillin, and organic anion-transporting polypeptide 1B3 (OATP1B3) inhibitors rifampicin and cyclosporin. Cell viability was established by trypan blue exclusion, annexin V staining, and a MTS assay. Caspase-3/7 activity was determined with Caspase-Glo assay, and cleaved caspase-3 was quantified by Western analysis. Cell number and colony-forming units were quantified after exposure to α-amanitin in primary CD34+ hematopoietic stem cells. In all cell lines, α-amanitin concentration-dependently decreased viability and mitochondrial activity. β-Amanitin was less toxic, but still significantly reduced viability. α-Amanitin increased caspase-3/7 activity by 2.8-fold and cleaved caspase-3 by 2.3-fold. Z-VAD(OH)-FMK significantly reduced α-amanitin-induced toxicity. In CD34+ stem cells, α-amanitin decreased the number of colonies and cells. The antidotes and OATP1B3 inhibitors did not reverse α-amanitin-induced toxicity. In conclusion, α-amanitin induces apoptosis in hematopoietic cells via a caspase-dependent mechanism.
摘要：
蛇毒蛇毒中毒占致命蘑菇中毒的大多数。最近,我们确定了血液毒性是天牛中毒的一个相关方面。在这项研究中,我们调查了鸡毒的主要毒素的影响，α-和β-amanitin，对体外造血细胞活力的影响。将造血细胞系暴露于α-amanitin或β-amanitin长达72小时，有或没有pan-caspase抑制剂Z-VAD（OH）-FMK，解毒剂N-乙酰半胱氨酸，水飞蓟宾,和青霉素,和有机阴离子转运多肽1B3（OATP1B3）抑制剂利福平和环孢菌素。通过锥虫蓝排除建立细胞活力，膜联蛋白V染色，和MTS分析。用Caspase-Glo测定法测定Caspase-3/7活性，和裂解的caspase-3通过Western分析定量。在暴露于原代CD34+造血干细胞中的α-amanitin后,定量细胞数量和集落形成单位。在所有细胞系中，α-amanitin浓度依赖性地降低了活力和线粒体活性。β-Amanitin毒性较小，但仍显著降低生存能力。α-Amanitin将caspase-3/7活性提高2.8倍，并将caspase-3裂解2.3倍。Z-VAD（OH）-FMK显着降低了α-amanitin诱导的毒性。在CD34+干细胞中，α-amanitin减少了集落和细胞的数量。解毒剂和OATP1B3抑制剂不能逆转α-amanitin诱导的毒性。总之,α-amanitin通过caspase依赖性机制诱导造血细胞凋亡。
