Acute Toxicological Impact Of Multiwall Carbon Nanotubes On Healthy Adult Mice

Carbon nanotubes have been attracted great attention due to its unique physical and chemical properties.As a new kind of nanomaterials,on one hand,carbon nanotubes might have potential applications in biomendical fields;on the other hand,because of the light weight,carbon nanotubes can easily spread in the air.Thus,it is imperative to investigate the effects of carbon nanotubes on biosystems.Correspondingly,the number of reports on the toxicity of carbon nanotubes has increased these days.However,few studies on systematic toxicity evaluation were reported to date.We first systematicly study the acute toxicity of intravenously injected carboxylated MWNTs on mice.OBJECTIVES1.To systematicly evaluate the toxicity of carboxylated MWNT on mice following intravenous injection.2.To compare the biological effects in vivo following intravenous injection between carboxylated MWNT with different diameters.METHODS1.Carboxylated multiwall carbon nanotubes preparationMulti-walled carbon nanotubes were treated by sonication and acid reflux to afford water-soluble nanotubes that contain acidic surface groups.2.Protein adsorption and BCA assayIn a typical protein adsorption experiment,0.2 mL MWNTs suspension(200ug/mL)was incubated with mice serum with a final concentration of 5%in PBS at 37℃for 2 h.Thereafter,the MWNTs were separated from the plasma by using centrifugation at 13000g for 8min and the supernatant was collected to perform a BCA assay according to the supplier's information.3.Animals and treatmentAnimal experiment was performed with compliance of the local ethics committee.The healthy BALB/C mice,about 18-22 g were supplied by the Experimental Animal Center,Shandong university. The animals were housed in clean polypropylene cages and maintained in an air-conditioned animal house at 20±2℃,50-70% relative humidity and 12-h light/dark cycle.The animals were provided with commercial mice diet and deionized water ad libitum. After acclimation for one week,the mice were randomly divided into four groups:Mice in group A were injected via the tail vain with 200ul of PBS containing 100ug 10nm-MWNT-COOH.Mice in group B were injected via the tail vain with 200ul of PBS containing 100ug 40nm-MWNT-COOH and 1 wt%Tween 80.For negtive control,the mice in group C were injected via the tail vain with 200ul of PBS and mice in group D were injected via the vain with 200ul of PBS containing 1%tween 80.After administration,the skin and fur changes,eye secretion,respiration and behavior patterns of the mice were observed.Special attention was paid on the clinical signs of toxicity including tremors,convulsions,salivation,nausea,vomiting, diarrhoea,lethargy,coma,etc.The body weights of mice were recorded every week after injection,1day,7days and 28days later, ten mice in each group were sacrificed separately and the blood was obtained from ophthalmic veins after.The heart,liver,spleen, kidneys,lungs,brain were collected.Two small blocks of the above organic tissues of both male and female mice for each group were kept in 10%formalin for histopathological examination.4.Biochemical assay of serumThe serum was obtained by centrifugation of the whole blood at 3000g for 15 min.The serum biochemical levels includingalanine aminotransferas(ALT),aspartate aminotransferase(AST),creatinine (CR),Blood Urea Nitrogen(BUN)were assayed by an automatic biochemical analyzer.5.Histopathological observationA small piece of heart,liver,kidney,spleen,lung,pancreas,stomach, and brain was fixed by 10%formalin and then embedded into paraffin, sectioned for 8-urn thick,and mounted on the glass microscope slides using standard histopathological techniques.The sections were stained with hematoxylineosin and examined by light microscopy.RESULTS:Surface protein adsorption adsorptionThe surface concentration of proteins adsorbed onto the particles from 5%mice plasma as determined by the micro-BCA assay. Compared with the MWNTs with larger diameters(40nm),MWNTs with smaller diameters(10nm)adsorbed larger amounts of proteins.Clinical observationsThe administration of the 10nm-MWNT-COOH and 40nm-MWNTs-COOH did not cause mortality in any of the treated groups.There were no signs of general clinical symptoms of toxicity or abnormal behavioral reactions.Body weightTreatment with MWNT-COOH for 4 weeks did not cause any adverse effects on growth because no statistically significant differences in the body weight gain were observed between the MWNT-COOH treated mice and control miceBiochemical assay of serumIn clinical biochemistry tests,no significant changes were observed in the MWNT-COOH-treated groups3.3 Pathological examinationA large number of 10nm-MWNT-COOH aggregates were found in the lungs of mice in group A at 1 day and 7 days after injection.A few 10nm-MWNT-COOH aggregates were cleared from the lungs after and many10nm-MWNT-COOH aggregates still retained in the lungs after 28 days.The 10nm-MWNT-COOH cleared from the lungs accumulated in the liver and could not be cleared from the liver within 28 days.On contrary,no 40nm-MWNT-COOH aggregate was found in the lungs of the mice in group B.Many 40nm-MWNT-COOH aggregate were found in mice liver 1 day after injection.The accumulation of 40nm-MWNT-COOH aggregate decreased with the exposure time.Both the two kinds of MWNT-COOH tended to undergo liver and spleen uptake and it is difficult for 10nm-MWNT-COOH to be cleared from the liver.MWNTs were localized into the kuppfer cells and were also found in the macrophages in the spleen.Scattered inflammation cells in stroma of myocardium were found in heart after 1 day and 7 days but heart were normal at 28 days after injection.The accumulations of MWNTs were still found at 28 days in some organs such as lungs and liver.However,no obviously histopathological change was observed.CONCLUSIONS:1.In comparison with 40nm-MWNT-COOH,10nm-MWNT-COOH tend to adsorbed more mice serum protein.2.c-10nm-MWNTs were mainly taken up by the mice lungs,liver and spleen.Although some c-10nm-MWNTs accumulations were cleared from the lungs,a great number of c-10nm-MWNTs were retained in the lungs at the relatively high accumulation levels over 28 days.The fact that the decrease of c-10nm-MWNTs accumulations and the increase of c-10nm-MWNTs accumulations in the liver suggested that the liver took up some c-10nm-MWNTs in the blood pool after it were cleared from the lungs.3.Both c-10nm-MWNTs and c-40nm-MWNTs tend to undergo RES uptake.4.We demostrated single administrations of high doses carboxylated MWNTs(5mg/kg)following intravenous injection did not lead to acute toxicity in BALB/C mice by the systematic toxicity evaluation...