Nanomaterials induce DNA-protein crosslink and DNA oxidation: A mechanistic study with RTG-2 fish cell line and Comet assay modifications.

Genotoxic effects of nanomaterials (NMs) have been controversially reported in literature, and the mode of action (MoA) via DNA oxidation is cited as the main damage caused by them. Evidence of nano-silver as a crosslinker has been previously reported by the present research team in an in vivo fish genotoxicity study. Thus, aiming to confirm the evidence about NMs as crosslinker agent, the present investigation elucidated the genotoxic potential of NMs and their genotoxic MoA through in vitro assay with RTG-2 cells line (rainbow trout gonadal) by exposure to nano-silver (PVP-coated) and nano-titanium. The types and levels of DNA damage were assessed by the Comet assay (standard alkaline, hOGG1-modified alkaline, and two crosslink-modified alkaline versions). It was demonstrated that the use of the standard alkaline Comet assay alone may inaccurately predict the genotoxicity of NMs since oxidative and crosslink DNA damages were also verified in RTG-2 cells when assessed by the modified versions of the alkaline protocol. More importantly, it was confirmed that both nano-silver and nano-titanium acted as DNA-protein crosslinkers through the Comet assay version with proteinase K. As both nano-silver and nano-titanium present a great risk to aquatic life, these findings reinforce the need of genotoxicity testing strategies that encompass the assessment of different types of DNA damage, in order to ensure an accurate prediction of the genotoxic potential of NMs.

Acute and long-term effects of trophic exposure to silver nanospheres in the central nervous system of a neotropical fish Hoplias intermedius.

Nanotechnologies are at the center of societal interest, due to their broad spectrum of application in different industrial products. The current concern about nanomaterials (NMs) is the potential risks they carry for human health and the environment. Considering that NMs can reach bodies of water, there is a need for studying the toxic effects of NMs on aquatic organisms. Among the NMs' toxic effects on fish, the interactions between NMs and the nervous system are yet to be understood. For this reason, our goal was to assess the neurotoxicity of polyvinylpyrrolidone coated silver nanospheres [AgNS (PVP coated)] and compare their effects in relation to silver ions (Ag+) in carnivorous Hoplias intermedius fish after acute and subchronic trophic exposure through the analysis of morphological (retina), biochemical (brain) and genetic biomarkers (brain and blood). For morphological biomarkers, damage by AgNS (PVP coated) in retina was found, including morphological changes in rods, cones, hemorrhage and epithelium rupture, and also deposition of AgNS (PVP coated) in retina and sclera. In the brain biomarkers, AgNS (PVP coated) did not disturb acetylcholinesterase activity. However, lowered migration of the DNA tail in the Comet Assay of blood and brain cells was observed for all doses of AgNS (PVP coated), for both acute and subchronic bioassays, and in a dose-dependent manner in acute exposure. Ag+ also reduced the level of DNA damage only under subchronic conditions in the brain cells. In general, the results demonstrated that AgNS (PVP coated) do not cause similar effects in relation to Ag+. Moreover, the lowered level of DNA damage detected by Comet Assay suggests that AgNS (PVP coated) directly interacts with DNA of brain and blood cells, inducing DNA-DNA or DNA-protein crosslinks. Therefore, the AgNS (PVP coated) accumulating, particularly in the retina, can lead to a competitive disadvantage for fish, compromising their survival.

Effects of realistic concentrations of TiO2 and ZnO nanoparticles in Prochilodus lineatus juvenile fish.

The impact of nanoparticles on fish health is still a matter of debate, since nanotechnology is quite recent. In this study, freshwater benthonic juvenile fish Prochilodus lineatus were exposed through water to three concentrations of TiO2 (0.1, 1, and 10 µg l(-1)) and ZnO (7, 70, and 700 µg l(-1)) nanoparticles, as well as to a mixture of both (TiO2 1 µg l(-1) + ZnO 70 µg l(-1)) for 5 and 30 days. Nanoparticle characterization revealed an increase of aggregate size in the function of concentration, but suspensions were generally stable. Fish mortality was high at subchronic exposure to 70 and 700 µg l(-1) of ZnO. Nanoparticle exposure led to decreased acetylcholinesterase activity either in the muscle or in the brain, depending on particle composition (muscle-TiO2 10 µg l(-1); brain-ZnO 7 and 700 µg l(-1)), and protein oxidative damage increased in the brain (ZnO 70 µg l(-1)) and gills (ZnO 70 µg l(-1) and mixture) but not in the liver. Exposed fish had more frequent alterations in the liver (necrosis, vascular congestion, leukocyte infiltration, and basophilic foci) and gills (hyperplasia and epithelial damages, e.g., epithelial disorganization and epithelial loss) than the control fish. Thus, predicted concentrations of TiO2 and ZnO nanoparticles caused detectable effects on P. lineatus that may have important consequences to fish health. But, these effects are much more subtle than those usually reported in the scientific literature for high concentrations or doses of metal nanoparticles.