Comparative Proteomics Analysis Of Pulmonary Toxic Effects Induced By Three Kind Of Typical Nanomaterials

Background:In recent years,as nanotechnology and materials science progressed by leaps and bounds,engineered nanomaterials have been mass-produced and widely applied.Simultaneously,people are increasingly exposed to various kinds of manufactured nanoparticles.Though the biological effects of some nanomaterials have been already assessed,the underlying mechanism is little understood.we speculated that protein molecules were involved in harmful effects of nanomaterials.So we plant to used proteomics technique to explore the toxicity mechanism of nanomaterials and to find indicative markers for its toxicity.Objective:In this paper,we aimed to:1) investigate the general toxicity of three types of nanomaterials;2) find differentially expressed proteins of injured lung with proteome between control and nanometerials gruops;3) further confirm differentially expressed protein by western blot,meanwhile,detecte the mRNA level of by RT-PCR.Methods:Nano-SiO₂,Nano-Fe₃O₄ and SWCNTs were respectively dispersed in normal sodium and characterized using TEM.49 Wistar rats were divided into 7 groups randomly including the control group,low and high dose groups of three nanomaterials.The Rats were exposed by intratracheal instillation once two days for 16 time,and then were killed after blood samples were collected by fossa orbitalis plexus venosus.We investigated lung,liver and kidney by pathological obversation.Biochemical indicator in serum,lactate dehydrogenas(LDH),total antioxid antcapacity (T-AOC),super oxid dismutase(SOD),malondialdeh-yde(MDA),interleukin-1(IL-1), interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) in bronchoalveolar lavage fluid(BALF) were detected.Two-dimensional Electrophoresis(2-DE)was performed to separate the proteins of Lung tissue.The 2-DE maps of nanometerials groups and control group were analyzed to find differentially expressed proteins,and the differential proteins were identified by MALDI-TOF mass spectrometry and database index.Then the expression level of identified,proteins transglen 2 was further confirmed by western blot,meanwhile,the mRNA level of the protein was detected by RT-PCR.Results:(1) Three nanomaterials produced rats'weight lose,but the renal and hepatic index had no significantly changes in every groups.(2) The observations of pathological analysis indicated small amounts hepatic cell adipose degeneration in the liver of rats,meanwhile,the fibrous tubercle and the matrix inflammation in lung tissue in nanometerials groups.kindey had no obvious change.(3) In rats'serum,aspartate transaminase(AST),alanine transaminase(ALT),alkalinephosphatase(ALP),uric acid (UA),creatinine(CREA) decreased,while blood urea nitrogen(BUN) increased in nanometerials groups.Albumin(ALB)and tatol protein(TP)had no obvious change.(4) The activities of T-AOC and SOD decreased while IL-6 concentration increased in nanometerials groups.In addition,LDH activities and MDA concentration increased in nanometerials groups in high dosage levels.The concentration of TNF-αincreased in 10mg.ml^-1 Nano-SiO₂ and 10mg.ml^-1 SWCNTs group.The toxic effect comparision among three nanomaterials were significantly distinct in the same dose.(5) Compared to the control group,17 protein spots were significantly altered in 2-DE gels of the nanometerials groups,they were related to metabolism,immunity,ion channel, oxidation stress,signal transduction and cytoskeleton.(6)The expression of transgelin 2 in lung were indeed increased in nanometerials groups tested by western blot,and this result further confirmed the result of 2-DE.The result of RT-PCR indicated that the mRNA level of transgelin2 in lung wasrespectively increased compared to the control group.Conclusions:Our results indicated that three typical nanomaterials which were instilled into lung through airway can bring damage to rat and cause some effect on the liver,kidney and lung.The possible routes maybe primarily ascribed to oxidative damage and inflammatory reaction.Meanwhile,comparative proteomics analysis demonstrated that the nanomaterials can make some proteins of lung alter uniformly.The comparative analysis suggestted that particle composition probably played a primary role in toxic effects of different nanoparticles.