Internalization and toxicological mechanisms of uncoated and PVP-coated cerium oxide nanoparticles in the freshwater alga Chlamydomonas reinhardtii

Due to the wide range of applications of cerium oxide nanoparticles (CeO2NPs), a risk assessment of their biological effects using environmentally relevant species becomes highly important. There are contradictory reports on the effects of CeO2NPs, which may be related to the use of different types of nanoparticles (NPs) and coatings. CeO2NPs may act as an oxidant causing toxicity or as an antioxidant able to scavenge free radicals. As a consequence of such complexity, the toxicological behaviour of these NPs is still poorly understood. Moreover, little is known about the internalization process of CeO2NPs in algae. There is evidence of CeO2NP-internalization by the green alga Chlamydomonas reinhardtii, but the mechanism and route of uptake are still unknown. In this study, we used uncoated and different polyvinylpyrrolidone (PVP)-coated CeO2NPs with the aim of identifying their toxicological mechanisms to C. reinhardtii and exploring their possible internalization. Our results showed that PVP coated-CeO2NPs significantly increased the formation of reactive oxygen species in exposed cells, indicating that oxidative stress is an important toxicity mechanism for these particles. Direct contact and damage of the cellular membrane was identified as the mechanism causing the toxicity of uncoated NPs. From experiments with endocytosis inhibitors, clathrin-dependent endocytosis was revealed as the main internalization route for all NPs. However, as uncoated CeO2NPs led to severe cellular membrane damage, direct passage of NPs through membrane holes could not be discarded. To our knowledge, this is the first report with evidence of direct linking between NP internalization and a specific endocytic pathway. The results presented here will help to unravel the toxicological mechanism and behaviour of CeO2NPs and provide input information for the environmental health and safety assessment of CeO2NPs.This research was supported by CTM2013-45775-C2-1,2-R and CTM2016-74927-C2-1,2-R grants from MINECO. NanoMILE (Grant Agreement no 310451 to EVJ & SMB) and the Endeavour Scholarship Scheme (Group B) (to SMB) are acknowledged.Peer reviewe