IN VITRO AND IN VIVO ASSESSMENT OF GLYPHOSATE IN NORMOXIA AND HYPOXIA CONDITIONS

Glyphosate (N-(phosphonomethyl) glycine) is a systemic and non-selective post-emergence foliar herbicide. It is today known as the most widely used herbicide worldwide. Because of the extensive use of glyphosate in agriculture, traces of this herbicide are nowadays found in soil, water, and air, as well as in food, becoming a growing concern for human health. A distinctive feature of some of these water environments, particularly those highly polluted, is the low water oxygen concentration. This leads to the development of hypoxic environments which represents a stress factor for the residing organisms. In zebrafish the adverse out- come pathway of the developmental toxicity of GLY and its underlying mechanisms remain still unclear. To date, little is still known about the combined effect of glyphosate and hypoxic conditions on the embryonal and larval forms found in water ecosystems and on in vitro model. Therefore, this study aims to assess the biochemical, histological and molecular changes of GLY-induced developmental toxicity in zebrafish embryos also to evaluate these behaviors associated with a hypoxic condition, chemically induced by cobalt chloride (CoCl2). The histological observation per- formed for each glyphosate concentration (25, 50, 75, 100 mg/mL) both in hypoxic and non-hypoxic conditions. The larvae showed major lesions in the liver and the intestine. To gain deeper insights into the role of hypoxia in the damage determined by Glyphosate, the presence of the toxic dam- age and inflammation markers have been evaluated in 100 mg/mL glyphosate-treated larvae both in normal and hypoxic conditions. To deeply investigate the effects of Hypoxia on glyphosate toxicity we decided to investigate low dose (50mg/L) of glyphosate in hypoxia condition. First we analyzed the mRNA and protein levels of Hif1a in all conditions. Regarding the expression levels of the major enzymes involved in the oxidative stress response, such as sods, catalase, gst. We observed a different modulation depending on the treatment. This first set of data allowed us to understand that all treatments were responding to oxidative stress, thus we decided to investigate also the enzymatic activity. As expected, the picture of enzymatic activity is also modulated by the treatments, indicating a response to oxidative stress. To better understand the effects of this enzymatic activity we performed the TBARs assay to evaluate the levels of lipid peroxidation products, which is one of the effects of ROS production. The only condition that was not able to counteract the increase of oxidative stress is the condition of glyphosate in hypoxia, suggesting a worsening effect of hypoxia on glyphosate toxicity. Finally to better dissect the effects of glyphosate in hypoxia conditions also in vitro models for the gut and liver have been used.