Zinc oxide nanoparticles (ZnONPs) are used in large quantities by the cosmetic, food and textile industries. Here we exposed Caenorhabditis elegans wild-type and a metal sensitive triple knockout mutant (mtl-1;mtl-2;pcs-1) to ZnONPs (0-50mg/L) to study strain and exposure specific effects on transcription, reactive oxygen species generation, the biomolecular phenotype (measured by Raman microspectroscopy) and key endpoints of the nematode life cycle (growth, reproduction and lifespan). A significant dissolution effect was observed, where dissolved ZnO constituted over 50% of total Zn within a two day exposure to the test medium, suggesting that the nominal exposure to pure ZnONPs represents in vivo, at best, a mixture exposure of ionic zinc and nanoparticles. Nevertheless, the analyses provided evidence that the metallothioneins (mtl-1 and mtl-2), the phytochelatin synthase (pcs-1) and an apoptotic marker (cep-1) were transcriptionally activated. In addition, the DCFH-DA assay provided in vitro evidence of the oxidative potential of ZnONPs in the metal exposure sensitive triple mutant. Raman spectroscopy highlighted that the biomolecular phenotype changes significantly in the mtl-1;mtl-2;pcs-1 triple knockout worm upon ZnONP exposure, suggesting that these metalloproteins are instrumental in the protection against cytotoxic damage. Finally, ZnONP exposure was shown to decrease growth and development, reproductive capacity and lifespan, effects which were amplified in the triple knockout. By combining diverse toxicological strategies, we identified that individuals (genotypes) housing mutations in key metalloproteins and phytochelatin synthase are more susceptible to ZnONP exposure, which underlines their importance to minimize ZnONP induced toxicity.
Keywords: C. elegans; CARS; Cd; Coherent anti-Stokes Raman spectroscopy; DCFH-DA; DLS; EDX; MT; Metallothionein; NGM; PBS; PC; PCA; Phytochelatin synthase; ROS; Raman spectroscopy; Reactive oxygen species; TEM; Zinc oxide nanoparticles; Zn; ZnONP; cadmium; dichlorodihydrofluoroscein diacetate; dynamic light spectroscopy; energy-dispersive X-ray spectroscopy; metallothionein; nematode growth medium; phosphate buffered saline; phytochelatin; principle component analysis; qRT-PCR; quantitative Real-Time polymerase chain reaction; reactive oxygen species; transmission electron microscopy; zinc; zinc oxide nanoparticles.
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