Graphene oxide disruption of homeostasis and regeneration processes in freshwater planarian Dugesia japonica via intracellular redox deviation and apoptosis

The aquatic system is a major sink for engineered nanomaterials released into the environment. Here, we assessed the toxicity of graphene oxide (GO) using the freshwater planarian Dugesia japonica, an invertebrate model that has been widely used for studying the effects of toxins on tissue regeneration and neuronal devel-opment. GO not only impaired the growth of normal (homeostatic) worms, but also inhibited the regeneration processes of regenerating (amputated) worms, with LC10 values of 9.86 mg/L and 9.32 mg/L for the 48-h acute toxicity test, respectively. High concentration (200 mg/L) of GO killed all the worms after 3 (regenerating) or 4 (homeostasis) days of exposure. Whole-mount in situ hybridization (WISH) and immunofluorescence analyses suggest GO impaired stem cell proliferation and differentiation, and subsequently caused cell apoptosis and oxidative DNA damage during planarian regeneration. Mechanistic analysis suggests that GO disturbed the antioxidative system (enzymatic and non-enzymatic) and energy metabolism in the planarian at both molecular and genetic levels, thus causing reactive oxygen species (ROS) over accumulation and oxidative damage, including oxidative DNA damage, loss of mitochondrial membrane integrity, lack of energy supply for cell dif-ferentiation and proliferation leading to retardance of neuron regeneration. The intrinsic oxidative potential of GO contributes to the GO-induced toxicity in planarians. These data suggest that GO in aquatic systems can cause oxidative stress and neurotoxicity in planarians. Overall, regenerated tissues are more sensitive to GO toxicity than homeostatic ones, suggesting that careful handling and appropriate decisions are needed in the application of GO to achieve healing and tissue regeneration.