Abstract
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.
Highlights
Environmental or occupational exposure to the herbicide Atrazine (ATR, 6-chloro-N2-ethyl-N4-isopropyl-1,3,5-triazine2,4-diamine), which is one of the most commonly used agricultural products worldwide, can produce toxic consequences in animals and humans [1,2,3]
ATR treatment produced a significant increase (P,0.01 versus CON) in transferase mediated dUTP nick end labeling (TUNEL) positivity which was reduced significantly by MEL (P, 0.01 versus ATR; Fig. 1B). These results show that ATR induced apoptosis in splenocytes, which was reversed by MEL
Autophagy plays an important role in the recovery from endoplasmic reticulum (ER) stress [17] we investigated whether ATR and MEL could modulate the key autophagic signals BECN-1, LC3B-II and p62/SQSTM1
Summary
Environmental or occupational exposure to the herbicide Atrazine (ATR, 6-chloro-N2-ethyl-N4-isopropyl-1,3,5-triazine2,4-diamine), which is one of the most commonly used agricultural products worldwide, can produce toxic consequences in animals and humans [1,2,3]. As a potent endocrine disruptor, ATR targets endocrine, reproductive and nervous systems [4,5,6]. It modulates immunity [7] due to the interaction between endocrine and immune systems [8]. ATR-induced suppression of immunity foreshadows the risk for contracting diseases. ATR induced immunotoxicity is manifested by a decline in the number of splenic dendritic cells, selected subpopulations of splenic lymphocytes, thymic T cell populations and Natural Killer cell activity along with an increase in early apoptosis and reduction in weights of spleen and thymus [2,10,11,12]. Fas-mediated apoptosis has been identified as a mechanism for ATR immunotoxicity in splenocytes of mice [13] though additional mechanisms for immunotoxicity may exist
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