Molecular effectors in the chronic exposure to arsenic as early and sensitive biomarkers in developing Rhinella arenarum toads

Abstract

Arsenic, a natural element of ecological relevance, is one of the most toxic elements present in various regions of the world. It can be found in natural water sources throughout Argentina in concentrations between 0.01 and 15 mg L–1. The Argentinean autochthonous toad Rhinella arenarum was selected to study the molecular mechanisms involved in the effects and response to the chronic As exposure along its embryonic and larval development. We evaluated the effects on MAPK signal transduction pathway and transcription factors c-FOS and c-JUN, and the regulation of the expression at protein levels of different antioxidant enzymes. Our results indicated that As is modulating the MAPK pathway, increasing MEK and ERK levels both in the nuclear and post-nuclear fraction along the embryonic development and mainly at the beginning of the larval stage. Through this pathway, As can upregulate transcription factors like c-FOS and c-JUN, impacting the antioxidant response of the exposed embryos and larvae through antioxidant enzymes and recycling of GSH. Arsenic triggered specifically the synthesis of antioxidant enzymes in exposed R. arenarum embryo and larvae. In particular, the expression levels of SOD, CAT and GST enzymes analyzed by Western blot showed a similar behavior to their enzymatic activities in our previous work. This fact suggests that not only the synthesis of these antioxidant enzymes but also their rapid degradation after inactivation would be regulated in response to ROS levels. Antioxidant enzymes may show dual responses of induction and inactivation followed by degradation depending on the levels of oxidative stress and impact on ROS targets when the exposure is sustained in time and intensity. We also performed a probability of exceedence analysis including our previous results to visualize a progression of the response in time and also established the best early-responding biomarkers at the lowest As concentrations. As a conclusion, the molecular biomarkers such as the MAPKs MEK and ERK and transcription factors c-FOS and c-JUN are early induced in the response of developing toad embryos exposed to very low As concentrations in water. The advantage of counting with molecular biomarkers early responding to low concentrations of As in a chronic exposure is that they may anticipate the irreversible damage at later developmental stages due to the constant oxidative challenge.