Biotoxicity,in Vivo Distribution And Acute Toxicological Mechanism Of Environmental Fe₃O₄ Nanoparticles

Background and objective:Environmental particles threaten human health,and tens of millions of people lose their lives every year due to their exposure.In recent years,the deposition of environmental magnetic particles represented by ferric tetroxide（Fe₃O₄）has been found many times in human organs,and this phenomenon has also attracted extensive attention from the academic community.Although studies have confirmed that environmental Fe₃O₄ is related to human diseases,there is still a lack of understanding of its biological toxicity,body distribution,and toxicological mechanisms.This paper takes environmental Fe₃O₄ as the research object.To understand its physical and chemical properties,multi-faceted characterization methods were used.By establishing models at multiple levels of cells,tissues,and animals,their biological toxicity,distribution,and metabolism can be evaluated.Through high-throughput sequencing analysis,the biological toxicity mechanism mediated by environmental Fe₃O₄ was further explored.Materials and methods:First of all,this paper extracts particulate matter from the waste gas of magnetic ore fuel combustion sites and vehicles and extracts Fe₃O₄ from the particulate matter through a self-made particulate matter screening device.Then,the physicochemical properties of environmental Fe₃O₄ were evaluated by a magnetic measurement system（VSM）,transmission electron microscope（TEM）,X-ray energy dispersive spectrometer（XPS）,and other equipment;and the biological properties of environmental Fe₃O₄in vitro were evaluated in combination with cytotoxicity and cellular uptake experiments.Toxicity.Furthermore,the in vivo distribution and in vivo toxicity of environmental Fe₃O₄ were explored using nuclear magnetic resonance（MRI）,physiology,behavior,biochemistry,and pathology.Finally,the biotoxicity mechanism mediated by environmental Fe₃O₄ was further explored through high-throughput proteomic analysis combined with bioinformatics methods.Results:The results of XPS and TEM show that the particles extracted in this study are Fe₃O₄.The morphology of environmental Fe₃O₄ is roughly round or square.At the same time,the magnetic properties of ambient Fe₃O₄particles（4.5 emu/g）in the VSM results are lower than those of the standard（more than 20 emu/g）.In the in vitro uptake experiment,the cellular uptake ratio of ambient Fe₃O₄ particles（51.6%）was higher than that of cells in the Fe₃O₄ standard group（5nm:30%;100nm:43.3%）and was statistically significant（p<0.05）.In the in vitro apoptosis experiment,the proportion of apoptotic cells in the environmental Fe₃O₄ group（35.5%）and the 5 nm Fe₃O₄ group（15.6%）was significantly different from the control group（6.27%）（p<0.05）.In in vitro proliferation experiments,the differences in cell viability between environmental Fe₃O₄ and 5 nm Fe₃O₄compared with the control and 100 nm Fe₃O₄ groups were statistically significant（p<0.05）and showed a significant dose-dependent manner.The MRI in vitro experiments showed that the relaxation efficiencies of ambient Fe₃O₄,5 nm Fe₃O_4,and 100 nm Fe₃O₄ were 0.5,6.5,and 21.1,respectively.MRI in vivo experiments showed that during blood exposure,the T2 value of environmental Fe₃O₄ was 25.3ms 1 hour after exposure,and its T2 value gradually decreased with time,and the lowest T2 value was 25.1ms on the third day after exposure;After that,its T2 value gradually increased with the prolongation of exposure time,and the above values were statistically significant compared with the control group（p<0.05）,and other organs such as brain and kidney did not change significantly.When exposed to the respiratory tract and gastrointestinal tract,not only did the T2 value in the liver decrease later than that in the blood exposure in the environmental Fe₃O₄ group but also the decrease in the T2 value in the liver was smaller than that in the blood exposure（p<0.05）.Compared with the in vivo phenomenon of environmental Fe₃O₄,the aggregation phenomenon in the 5 nm Fe₃O₄ standard group（the lowest T2 value at blood exposure was 22.6ms）was about the same.The difference is that although the 100nm Fe₃O₄ standard group had the lowest T2 value of 23.6ms during blood exposure,the aggregation phenomenon caused by respiratory exposure and gastrointestinal exposure appeared a little later than that of the other two groups.In the physiological experiment,at 24 hours after blood injection,the mean values of the environmental Fe₃O₄ group and the 5 nm Fe₃O₄ group were 701 times/min and 651 times/min,respectively,compared with 485times/min in the control group,a statistically significant difference（p<0.05）.In behavioral experiments,mice in the environmental Fe₃O₄group and the 5 nm Fe₃O₄ group spent 120s and 117s on the rotarod 24hours after receiving blood exposure,respectively,compared to the control group of 222s,a statistically significant difference became shorter（p<0.05）.In the biochemical experiments,the content of total bilirubin in the environmental Fe₃O₄ and 5 nm Fe₃O₄ groups were 147umol/L and121umol/L,respectively,and the average values of alanine aminotransferase were 97U/L and 93U/L,respectively,which were comparable to the control group.There is a statistically significant difference（p<0.05）.The results of pathological Prussian blue showed that the Fe contents of environmental Fe₃O₄,5 nm Fe₃O_4,and 100 nm Fe₃O₄ in the liver were 1.54%,2%,and 1.72%,respectively,and the Fe accumulations in each group were significantly higher than those in other organs（p<0.05）.The H&E results showed that the mean ratios of inflammatory cell infiltration in the liver in the environmental Fe₃O₄ group and the 5 nm Fe₃O₄ group were 3.9%and 4.7%,respectively,compared with 1.2%and 1.4%in the control group and the 100 nm Fe₃O₄ group.Statistics There were differences in learning（p<0.05）.In the proteomic analysis,828 proteins were differentially expressed in the environmental Fe₃O₄ group,of which the number of up-regulated proteins was 287 and the number of down-regulated proteins was 541,which was much higher than the number of differential proteins in other groups（p<0.05）.GO analysis showed that the differential proteins in the environmental Fe₃O₄ group were mainly related to the acute response of the organism and the molecules in the metabolic pathway（p<0.05）.KEGG analysis showed that differential proteins in the environmental Fe₃O₄ group were related to cellular metabolism,chemical carcinogenesis,and glutathione metabolic pathways（p<0.05）.IPA analysis showed that the differential proteins in the environmental Fe₃O₄ group were related to body metabolism,liver function,and cancer（p<0.05）.The changes of differential proteins in the Fe₃O₄ group were verified by enzyme-linked immunosorbent assay,which was consistent with the results of bioinformatics analysis.Conclusion:The physical and chemical properties of environmental Fe₃O₄ are different from those of Fe₃O₄ in nature and the laboratory.They are not only widely distributed in the body,and their liver aggregation is the most significant in several important organs,but also have certain biological toxicity.The mechanism of toxicity is mainly related to the activation of oxidative stress and liver enzyme metabolism disorders.