Institute of Nuclear Medicine and Allied Sciences, Brig. S.K. Mazumdar Road, Delhi-110054, India.
Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), New Delhi 110062, India.
Toxicology. 2020 Jan 15;429:152324. doi: 10.1016/j.tox.2019.152324. Epub 2019 Nov 1.
Ochratoxin A (OTA) is known to induce nephro-toxicity via induction of cellular redox homeostasis perturbation, mitochondrial hyperpolarisation and depolarization, protein synthesis inhibition leading to apoptosis. In the present study, protective efficacy of N-Acetyl-L-Tryptophan (NAT) against OTA induced toxicity was evaluated using Human Embryonic Kidney (HEK-293) cells. Cells were treated with NAT (0-200 μg/ml) before OTA treatment (0-20 μg/ml) and protective efficacy of NAT was evaluated using MTT and SRB assay. OTA-induced intracellular ROS generation and its inhibition by NAT (2.5 μg/ml) pre-treatment was evaluated using 2',7'-dichlorodihydrofluorescein diacetate (HDCFDA) probe. Effects of NAT pre-treatment on OTA treated cells were also evaluated in terms of cell cycle perturbations and mitochondrial membrane potential disturbance using flowcytometry. Results of the study demonstrated significant (∼89 % cell growth in comparison to 50% in OTA alone group; P < 0.05) protection by NAT to the HEK-293 cells against OTA mediated cell death in terms of cell viability. Further, significant reduction in ROS levels and mitochondrial membrane potential disturbance was also observed in NAT pre-treated plus OTA cells as compared to only OTA treated cells. Significant (p < 0.05) arrest in G0 and S phase of cell cycle was observed in OTA treated cells that was found to be inhibited by NAT pre-treatment to OTA treated cells. Also, molecular docking analysis demonstrated higher probability of NAT to bind with OTA binding pocket on phenylalanyl t-RNA synthetase, resulting in inhibition of OTA incorporation in the newly synthesized peptides and thus may ameliorate OTA induced protein synthesis inhibition. Conclusively, present study suggested that NAT offers protection against OTA toxicity in HEK-293 cells by counterbalancing oxidative stress, cell cycle regulation, mitochondrial membrane potential stabilization, protein synthesis inhibition and cell death retardation.
赭曲霉毒素 A（OTA）通过诱导细胞氧化还原平衡紊乱、线粒体超极化和去极化、蛋白质合成抑制导致细胞凋亡而导致肾毒性。在本研究中，使用人胚肾（HEK-293）细胞评估 N-乙酰-L-色氨酸（NAT）对 OTA 诱导的毒性的保护作用。在 OTA 处理（0-20μg/ml）之前，用 NAT（0-200μg/ml）处理细胞，并使用 MTT 和 SRB 测定法评估 NAT 的保护作用。使用 2'，7'-二氯二氢荧光素二乙酸酯（HDCFDA）探针评估 OTA 诱导的细胞内 ROS 生成及其被 NAT（2.5μg/ml）预处理的抑制作用。还通过流式细胞术评估 NAT 预处理对 OTA 处理细胞的细胞周期扰动和线粒体膜电位干扰的影响。研究结果表明，与仅用 OTA 处理组的 50%相比，NAT 对 HEK-293 细胞具有显著的保护作用（∼89%的细胞生长；P<0.05），可以防止 OTA 介导的细胞死亡。此外，与仅用 OTA 处理的细胞相比，NAT 预处理加 OTA 细胞中的 ROS 水平和线粒体膜电位干扰也显著降低。在 OTA 处理的细胞中观察到细胞周期的 G0 和 S 期显著（p<0.05）停滞，该停滞被发现可被 NAT 预处理抑制。此外，分子对接分析表明，NAT 与苯丙氨酸 t-RNA 合成酶上的 OTA 结合口袋结合的可能性更高，从而抑制 OTA 掺入新合成的肽中，从而可能减轻 OTA 诱导的蛋白质合成抑制。综上所述，本研究表明，NAT 通过平衡氧化应激、细胞周期调节、线粒体膜电位稳定、蛋白质合成抑制和细胞死亡延缓，为 HEK-293 细胞提供对 OTA 毒性的保护作用。
