Sharma Shweta, Sarkar Jayanta, Haldar Chandana, Sinha Sudhir
Biochemistry Division, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India.
Biochemistry Division, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India; Academy of Scientific and Innovative Research, New Delhi, India.
PLoS One. 2014 Sep 26;9(9):e108602. doi: 10.1371/journal.pone.0108602. eCollection 2014.
Exposure to the herbicide Atrazine (ATR) can cause immunotoxicity, apart from other adverse consequences for animal and human health. We aimed at elucidating the apoptotic mechanisms involved in immunotoxicity of ATR and their attenuation by Melatonin (MEL). Young Swiss mice were divided into control, ATR and MEL+ATR groups based on daily (x14) intraperitoneal administration of the vehicle (normal saline), ATR (100 mg/kg body weight) and MEL (20 mg/kg body weight) with ATR. Isolated splenocytes were processed for detection of apoptosis by Annexin V-FITC and TUNEL assays, and endoplasmic reticulum (ER) stress by immunostaining. Key proteins involved in apoptosis, ER stress and autophagy were quantified by immunoblotting. ATR treatment resulted in Fas-mediated activation of caspases 8 and 3 and inactivation of PARP1 which were inhibited significantly by co-treatment with MEL. MEL also attenuated the ATR-induced, p53 independent mitochondrial apoptosis through upregulation of E2F-1 and PUMA and suppression of their downstream target Bax. An excessive ER stress triggered by ATR through overexpression of ATF-6α, spliced XBP-1, CREB-2 and GADD153 signals was reversed by MEL. MEL also reversed the ATR-induced impairment of autophagy which was indicated by a decline in BECN-1, along with significant enhancement in LC3B-II and p62 expressions. Induction of mitochondrial apoptosis, ER stress and autophagy dysregulation provide a new insight into the mechanism of ATR immunotoxicity. The cytoprotective role of MEL, on the other hand, was defined by attenuation of ER stress, Fas-mediated and p53 independent mitochondria-mediated apoptosis as well as autophagy signals.
除了对动物和人类健康产生其他不良后果外，接触除草剂阿特拉津（ATR）还会导致免疫毒性。我们旨在阐明ATR免疫毒性所涉及的凋亡机制以及褪黑素（MEL）对其的减轻作用。将年轻的瑞士小鼠根据每日（共14天）腹腔注射溶剂（生理盐水）、ATR（100mg/kg体重）和与ATR联用的MEL（20mg/kg体重）分为对照组、ATR组和MEL + ATR组。分离的脾细胞通过膜联蛋白V - FITC和TUNEL检测进行凋亡检测，并通过免疫染色检测内质网（ER）应激。通过免疫印迹对凋亡、ER应激和自噬相关的关键蛋白进行定量。ATR处理导致Fas介导的半胱天冬酶8和3激活以及PARP1失活，而与MEL共同处理可显著抑制这些作用。MEL还通过上调E2F - 1和PUMA并抑制其下游靶点Bax，减轻了ATR诱导的、p53非依赖性的线粒体凋亡。ATR通过ATF - 6α、剪接的XBP - 1、CREB - 2和GADD153信号的过表达引发的过度ER应激被MEL逆转。MEL还逆转了ATR诱导的自噬损伤，这表现为BECN - 1下降，同时LC3B - II和p62表达显著增强。线粒体凋亡、ER应激和自噬失调的诱导为ATR免疫毒性机制提供了新的见解。另一方面，MEL的细胞保护作用表现为减轻ER应激、Fas介导的和p53非依赖性的线粒体介导的凋亡以及自噬信号。
