Quantitative Proteomic Analysis to Understand the Mechanisms of Zinc Oxide Nanoparticle Toxicity to <i>Daphnia pulex</i> (Crustacea: Daphniidae): Comparing with Bulk Zinc Oxide and Zinc Salt

The widespread use of zinc oxide nanoparticles (ZnO NPs) has resulted in their release to the environment. There has been concern about the ecotoxicity of ZnO NPs, but little is known about their toxic mechanisms. In the present study, we conducted acute toxicity tests to show that ZnO NPs are more toxic to the freshwater crustacean Daphnia pulex compared to bulk ZnO or ZnSO4·7H2O. To provide an integrated and quantitative insights into the toxicity of ZnO NPs, we conducted isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis, which detected 262, 331, and 360 differentially expressed proteins (DEPs) in D. pulex exposed to ZnO NPs, bulk ZnO, and ZnSO4·7H2O, respectively. Among the DEPs, 224 were shared among the three treatments. These proteins were related to energy metabolism, oxidative stress, and endoplasmic reticulum stress. The three forms of Zn all caused D. pulex to downregulate Chitinase expression, disrupt Ca2+ homeostasis, and reduce expression of digestive enzymes. Nevertheless, 29 proteins were expressed only in the ZnO NP treatment. In particular, histone (H3) and ribosomal proteins (L13) were obviously influenced under ZnO NP treatment. However, increased expression levels of h3 and l13 genes were not induced only in ZnO NP treatment, they were sensitive to Zn ions under the same exposure concentration. These results indicate that the three zinc substances have a similar mode of action and that released zinc ions are the main contributor to ZnO NP toxicity to D. pulex under a low concentration. Further investigation is needed to clarify whether a small proportion of DEPs or higher bioavailability cause ZnO NPs to be more toxic compared to bulk ZnO or ionic zinc