Menotti Jean, Alanio Alexandre, Sturny-Leclère Aude, Vitry Sandrine, Sauvage Félix, Barratt Gillian, Bretagne Stéphane
Institut Pasteur, CNRS, Molecular Mycology unit, URA3012 Paris, France; Laboratory of Parasitology-Mycology, Saint-Louis Lariboisière Fernand Widal hospitals, Assistance Publique-Hôpitaux de Paris (AP-HP), Paris, France; Paris-Diderot, Sorbonne Paris Cité University, Paris, France; Laboratory of Parasitology-Mycology, Croix-Rousse Hospital, Hospices Civils de Lyon, Université Claude Bernard - Lyon 1, Lyon, France.
Institut Pasteur, CNRS, Molecular Mycology unit, URA3012 Paris, France; Laboratory of Parasitology-Mycology, Saint-Louis Lariboisière Fernand Widal hospitals, Assistance Publique-Hôpitaux de Paris (AP-HP), Paris, France; Paris-Diderot, Sorbonne Paris Cité University, Paris, France.
Toxicol Appl Pharmacol. 2017 Nov 1;334:18-23. doi: 10.1016/j.taap.2017.08.017. Epub 2017 Sep 1.
Aerosolized liposomal amphotericin B (L-AmB) has been investigated as prophylaxis against invasive aspergillosis. However, the clinical results are controversial and some trials suggest that toxicity could be a limitation for wider use. Our aim was to assess the dynamics of cell toxicity induced in a human alveolar epithelial cell line (A549) after exposure to L-AmB (50 to 400μg/ml) or amphotericin B deoxycholate (D-AmB; 50 to 200μg/ml) by monitoring real-time A549 cell viability using an impedance-based technology. Results were expressed as cell index values integrating cell adhesion, proliferation, and survival. In parallel, the gene expression of proinflammatory cytokines was quantified at 6 and 24h after drug addition by real-time RT-PCR on cell lysates. No sustained reduction of cell indexes was observed with L-AmB or empty liposomes, even at 400μg/ml. Only the highest concentration tested of L-AmB (400μg/ml) yielded transient significant 6-fold and 4-fold induction of TNF-α and IL-8 mRNAs, respectively. In contrast, D-AmB induced a decrease in cell indexes and only the 50μg/ml concentration of D-AmB was followed by cell recovery, higher concentrations leading to cell death. Significant 4-fold, 7-fold and 3-fold inductions of TNF-α, IL-8 and IL-33 mRNAs were also observed at 6h with 50μg/ml of D-AmB. In conclusion, continuous cell impedance measurement showed no toxicity on overall cellular behavior although a slight proinflammatory cytokine expression is possible after L-AmB challenge. Real-time kinetics of cell impedance is an interesting tool for initial screening of cell toxicity.
雾化脂质体两性霉素B（L-AmB）已被研究用于预防侵袭性曲霉病。然而，临床结果存在争议，一些试验表明毒性可能是其更广泛应用的限制因素。我们的目的是通过使用基于阻抗的技术监测人肺泡上皮细胞系（A549）在暴露于L-AmB（50至400μg/ml）或两性霉素B脱氧胆酸盐（D-AmB；50至200μg/ml）后的细胞毒性动态变化。结果以整合细胞黏附、增殖和存活的细胞指数值表示。同时，在添加药物后6小时和24小时，通过对细胞裂解物进行实时逆转录聚合酶链反应（RT-PCR）定量检测促炎细胞因子的基因表达。即使在400μg/ml时，L-AmB或空脂质体也未观察到细胞指数的持续降低。仅测试的最高浓度L-AmB（400μg/ml）分别短暂显著诱导肿瘤坏死因子-α（TNF-α）和白细胞介素-8（IL-8）mRNA增加6倍和4倍。相比之下，D-AmB导致细胞指数下降，只有50μg/ml浓度的D-AmB后细胞恢复，更高浓度则导致细胞死亡。在6小时时，50μg/ml的D-AmB也显著诱导TNF-α、IL-8和白细胞介素-33（IL-33）mRNA分别增加4倍、7倍和3倍。总之，连续细胞阻抗测量显示对整体细胞行为无毒性，尽管L-AmB刺激后可能有轻微的促炎细胞因子表达。细胞阻抗的实时动力学是初步筛选细胞毒性的有趣工具。
