Formation Mechanism And Toxicity Effect Of Nanoparticles Derived From Grilled Fish

Grilled fish,as a traditional food with attractive texture and flavors,has been favored by consumers for thousands of years.In the process of grilling,due to the high temperature,it is possible to produce carbonaceous nanostructures through a series of complex spontaneous chemical reactions.In the formation of these nanoparticles(NPs),the factors such as grilling materials,temperature and time will affect the structure of NPs,thus affecting their biological activities.Pike eel(Muraenesox cinereus)and turbot(Scophthalmus maximus L.)with two different nutrient compositions as raw materials were studied.The physical and chemical properties,formation mechanism and toxicity mechanism of NPs derived from these two kinds of grilling fish were exploredCarbon dots(CDs)and onion-like carbon nanoparticles(OCNPs)were extracted from grilled pike eel and grilled turbot,respectively.CDs are spherical and monodispersed,with a narrow size distribution in the range of 1.5 ~ 3.5 nm.The OCNPs have a strong aggregation and crystallization structure with a shell thickness of 3 ~ 5 nm and a spacing of 0.33 nm.Both of CDs and OCNPs mainly contained C,N,O and H elements with functional groups of hydroxyl,carboxyl,amide bonds and amino groups on the surface.Furthermore,ultraviolet(UV)light could excite both CDs and OCNPs to emit strong sapphire luminescence with excitation-dependent emission behavior,indicating different types of defect structure of their surface.The physicochemical properties of NPs from pike eel and turbot grilled at different temperature were compared and analyzed.The formation of NPs during the grilling pike eel is believed to go through a process of pyrolysis,nucleation and carbonization and so on.N content and sp2 structure increased gradually with the increase of grilling temperature.Likewise,in the grilling process,the turbot sections changed into relatively large size cluster of polymers by heat shrinkage and pyrolysis.Crystal lattice structure emerged for the OCNPs along with the increase of heating temperature possibly due to the enhanced oxidation reaction.Finally,the spherical carbon NPs with the lattices were formed under the action of carbonization reaction.A series of toxicity experiments were carried out to investigate the safety of different types of NPs in grilled fish.As for in vitro experiments,mouse osteoblasts cells(MC3T3-E1)were selected as model subjects,and incubated with CDs and OCNPs for 24 h,respectively.The results showed both CDs and OCNPs could enter into MC3T3-E1 cells,and evenly distribute in the cytoplasm,without any obvious effects on the MC3T3-E1 cell morphology.When the concentration of CDs or OCNPs was 20 mg/m L,the cell viability and the integrity of the cell membrane did not change significantly.However,the ROS level was significantly increased,which could lead to oxidative stress response.In addition,hemolysis rates of both CDs and OCNPs were less than 6% for in vitro hemolysis test,which did not cause serious hemolysis.As for in vivo tests,danio rerio was selected as the model organism for the short-term and long-term toxicity of CDs and OCNPs.For danio rerio,96 h LC50 values of CDs and OCNPs were 321.951 mg/L and 212.431 mg/L,respectively.In the subacute toxicity experiments,the exposure of CDs with a concentration of 65 mg/L for 14 days,and OCNPs at 30 ~ 40 mg/L for 10 days caused a significant increase of ROS in danio rerio.Oxidative stress promoted the activities of superoxide dismutase(SOD)and glutathion peroxidase(GSH-Px).Meanwhile,due to the excessive ROS,the antioxidant and detoxifying enzymes system was destroyed,the activity of both peroxidase(POD)and catalase(CAT)showed a downward trend.Besides,the oxidative damage induced lipid and protein peroxidation with the increase of malondialdehyde and protein carbonyl contents.In the long-term toxicity test,the danio rerio was exposed to different concentrations of CDs and OCNPs for 30 days.The only significant increase of ROS levels was found in the high concentration group of CDs(30 mg/L)and OCNPs(10 mg/L),and the activity of antioxidant enzymes were inhibited significantly with the protein and lipid oxidative damage.To further understand the metabolic processes of CDs and OCNPs in the danio rerio,the in vitro degradation experiment was carried out using PBS buffer as simulation reagent.After CDs and OCNPs being degraded in PBS solution for 30 days,the hydrolyzation was enhanced leading the formation of more hydroxyl groups on their surface,which could enhance the water solubility.The increasing solubility is helpful for excretion of NPs thus reducing their influence to the body.