| [Objective] By the Ames test, the comet test, the micronucleus assay and the sperm deformity test, from gene level to DNA level to chromosome level, the genotoxicity induced by alumina nanoparticles at different size was evaluated comprehensively, and the mechanism of action of alumina nanoparticles was explored.[Methods]1. Bacterial reverse mutation (Ames)assay(1) Characterization of the alumina nanoparticles (13nm and50nm) was done before the initiation of the test,image-pro plus image analysis software for size analysis and nano-ZS90nano-size instrument for particle potential.(2) Ames assay The bacterial reverse mutation (Ames) test with Salmonella typhimurium TA97, TA98, TA100, TA102strains, with and without the S9mixture were conducted. Alumina nanoparticles (13nm and50nm) and aluminium oxide-micro were selected. Use a wide range of concentration0.5,5,50,500,5000μg/plate, with negative control group, solvent group and positive control group.(3) Oxidative stress-response systems Glutathiose (GSH), superoxide dismutase (SOD), Malondialdehyde (MDA) and total anti-oxidative capacity (T-AOC) were used to assess oxidative stress-response systems.2. Comet test(1) The determination of IC502×104cells/ml were seeded in a six-well dish. After attachment, the culture medium were exchanged with fresh one containing different concentrations of alumina nanoparticles (13nm and50nm) and aluminium oxide-micro (10μm), at the final concentration0,1.5,3.0,6.2,12.5,25,50,100μg/ml, eight groups, each concentration repeated for three times. After treatment for24h, the culture medium were removed, cells were washed twice with D-Hank s solution, digested with0.25%trypsin, and done into single cell suspension. Calculate survival cells with inverted microscope, average the cells, then determined the inhibition rate, repeated three times, and calculated IC50of different size alumina nanoparticles with Probit method.(2) Cy5.5fluorescent labeled nanoparticles into the cells The CHL cells were treated with alumina nanoparticles (13nm and50nm) and aluminium oxide-micro (10μm) at the final concentration30μg/ml. After treated for3hours, Hoechst fluorescent dye was added to the culture medium to a final concentration of5μg/ml, then the cells were washed twice with the D-Hank’s solution after15min, at last the entry of alumina nanoparticles into cells was observed under a fluorescence microscope.(3) Single cell gel electrophoresisi (SCGE) Alumina nanoparticles (13nm and50nm) and aluminium oxide-micro (10μm) were selected. Use three groups:blank control group, low-dose (15μg/ml), medium-dose (30uμ/ml), high-dose (60μg/ml) and the positive control group (mitomycin C0.5μg/ml). After treated for12,24and48h, the DNA damage were detected by SCGE and evaluated with the Oliver tail.(4) Cellular oxidative stress-response systems Cellular oxidative stress system were assessed with glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA) and total antioxidant capacity (T-AOC).3. Mice bone marrow micronucleus test50male and50female ICR mice were divided into11groups,5in each group, alumina nanoparticles (13nm and50nm) and aluminium oxide-micro (10μm) were selected. Use three groups:blank control group, low-dose (300mg/kg), medium-dose (600mg/kg), high-dose (1200mg/kg) and the positive control group (cyclophosphamide40mg/ml) by intraperitoneal injection. Bone marrow were smearsed and micronucleus frequencey were calculated.4. Mice sperm abnormality test(1) Mice sperm abnormality test55male ICR mice were divided into11groups,5in each group, alumina nanoparticles (13nm and50nm) and aluminium oxide-micro (10μm) were selected. Use three groups:blank control group, low-dose (300mg/kg), medium-dose (600mg/kg), high-dose (1200mg/kg) and the positive control group (cyclophosphamide40mg/ml), intraperitoneal injection for5days. At the35days, the mice were dislocatedly killed and the epididymis, testis were taken and sperm abnormality rate were determined.(2) Testicular pathomorphology Left testis were fixed in4%neutral formalin solutioin for24h, then dehydrated, transparent, embedded in paraffin, sliced (slice thickness of about4mm), then follow-up HE staining, microscopic examination.(3) Oxidative stess-response system in mice testicular Oxidative stress-response system was assessed with glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA) and total antioxidant capacity (T-AOC).[Results]1. Ames test(1) Charaterization Size of alumina nanoparticles were20.9±9.5nm and112.4±9.1nm, respectively. Zeta potential were49.4±2.2mV and44.3±9.1mV, respectively. (2) Ames testIn the Reverse mutation assay of alumina nanoparticles (13nm and50nm) and aluminium oxide-micro (10μm) inactivated plate incorporation test, the number of revertant colonies of the strains induced by positive control group increased significantly more twice than the blank control group, and the difference was statistically significant (P<0.01), the revertant colonies of solvent control group were less twice than the blank control group. The revertant colonies of the strains induced by alumina nanoparticles (13nm and50nm) and aluminium oxide-micro (10μm) were less twice than the blank control group.In the Reverse mutation assay of alumina nanoparticles (13nm and50nm) and aluminium oxide-micro (10μm) activated plate incorporation test, the revertant colonies of the strains induced by positive control group increased, in particular strains TA97, TA98, TA100, more twice than the blank control group, and the difference was statistically significant (P<0.01), the revertant colonies of solvent control group were less twice than the blank control group. The revertant colonies of the strains induced by alumina nanoparticles (13nm and50nm) and aluminium oxide-micro (10μm) were less twice than the blank control group.(3) Bacterial oxidative-stress response system With and without liver S9mixture, the GSH, SOD, MDA, T-AOC induced by alumina nanoparticles (13nm and50nm) and aluminium oxide-micro (10μm), compare with the blank control group, difference were not statistically significant (P>0.05).2. the comet assay(1) IC50Calculated with the probit method, the IC50of alumina nanoparticles (13nm) was56.75μg/ml, the IC50of alumina nanoparticles (50nm) was44.06μg/ml, the IC50of aluminium oxide-micro (10μm) was64.27μg/ml. Based on the above, in order to facilitate comparison between the various particle size groups, uniform doses of exposure dose was selected:the high-dose60μg/ml, the medium-dose30μg/ml, low-dose15μg/ml.(2) The CHL cell nucleus presented a uniform blue dye fluorescence in fluorescent Hoechst staining and cy5.5fluorescent labeled alumina nanoparticles red particles. Merge Hoechst stainingf figures into fluorescent labeled figures, and figures showed more fluorescent labeled nanoparticles into the cytoplasm, less aluminium oxide-micro.(3)Single cell agarose gel electrophoresis The DNA of comet head of blank control group was dense, and no obvious tail or the tail was very short. The DNA of comet head of each exposure group was strong brightness, The DNA of tail broom-like tail composed by DNA fragments was less than the DNA of head in the fluorescence intensity. Dose-dependent effect: compared with the conrol group, the Oliver tail moment exposure to12,24and48h, with the increasing exposure dose, the different size groups of low-, medium-and high-dose groups increased significantly, the difference with the high-dose was statistically significant (P<0.05, P<0.01). Time-dependent effect:alumina nanoparticle (13n m) group,a lumina nanoparticle (50nm) group and aluminium oxide-micro (10μm) group Oliver tail moment, with prolonged exposure, there was an increasing trend, compared with the12and24h, the difference of Oliver tail moment exposure to48h was statistically significant (P<0.05), compared with the12h, the difference of Oliver tail moment exposure to24h was statistically significant(P<0.05). Size-dependent effect:exposed to48h, compared with the alumina nanoparticle (13nm) group and aluminium oxide-micro (10μm) group, the difference between the alumina nanoparticle (50nm) group was statistically significant(P<0.05).(4) Cell oxidative stress levels The results of GSH analyzed showed that dose-dependent effect:when exposed to24and48h, compared with the conrol group, with the increasing exposure dose, the GSH of different size groups of low-, medium-and high-dose groups decreased significantly, the difference with the high-dose was statistically significant (P<0.05, P<0.01). Time-dependent effect:with prolonged exposure, in GSH of alumina nanoparticle (50nm) group there was a decreasing trend, compared with the12and24h, the difference of GSH exposure to48h was statistically significant (P<0.05), compared with the24h, the difference of GSH exposure to12h was not statistically significant (P>0.05). Size-dependent effect:exposed to48h, compared with the alumina nanoparticle (13nm) group and aluminium oxide-micro (10μm) group, the difference between the alumina nanoparticle (50nm) group was statistically significant (P<0.05).The results of SOD analyzed showed that dose-dependent effect:when exposed to12and24h, compared with the conrol group, with the increasing exposure dose, the SODof the alumina nanoparticle (13nm) group and aluminium oxide-micro (10μm) group of low-, medium-and high-dose groups decreased significantly, the difference with the high dose was statistically significant (P<0.05, P<0.01). Time-dependent effect:exposed to48h, the SOD of different size groups of low-, medium-and high-dose groups decreased significantly, compared with the conrol group, the difference with the high-dose was statistically significant (P<0.01). Size-dependent effect: with prolonged exposure, in SOD of different size groups there was a decreasing trend, compared with the12h, the difference of SOD exposure to48h was statistically significant (P<0.01). When exposed to48h, compared with the alumina nanoparticle (13nm) group and aluminium oxide-micro (10μm) group, the difference between the alumina nanoparticle (50nm) group was statistically significant (P<0.05), compared with the aluminium oxide-micro (10μm) group, the difference beteen the alumina nanoparticle (13nm) group was statistically significant(P<0.05).The results of MDA analyzed showed that dose-dependent effect:when exposed to12,24and48h, compared with the conrol group, with the increasing exposure dose, the MDA of different size groups of low-, medium-and high-dose groups increased significantly, the difference with the medium-and high dose groups of alumina nanoparticle (13nm and50nm) groups and aluminium oxide-micro (10μm) group was statistically significant (P<0.01). Time-dependent effect:with prolonged exposure, there was an increasing trend, in MDA of different size groups. In alumina nanoparticle (13nm) group, the difference between12h and24,48h was statistically significant (P<0.05). In alumina nanoparticle (50nm) group and aluminium oxide-micro (10μm) group, the difference between48h and24,12h was statistically significant (P<0.05), the difference between12h and24h was statistically significant (P<0.05). Size-dependent effect:exposed to24h, the difference between the the alumina nanoparticle (50nm) group and alumina nanoparticle (13nm) group, aluminium oxide-micro (10μm) group was statistically significant (P<0.05, P<0.01). Exposed to48h the difference between the alumina nanoparticle (13nm and50nm) groups and aluminium oxide-micro (10μm) group was statistically significant (P<0.05), the difference between the alumina nanoparticle (13nm) group and alumina nanoparticle (50nm) group was statistically significant (P<0.05).The results of T-AOC analyzed showed that dose-dependent effect:when exposed to24and48h, compared with the conrol group, with the increasing exposure dose, the T-AOC of different size groups of low-, medium-and high-dose groups decreased significantly, the difference with the medium-and high-dose was statistically significant (P<0.01). Time-dependent effect:with prolonged exposure, in T-AOC of the alumina nanoparticles (13nm and50nm) there were a decreasing trend, compared with the12h, the difference of T-AOC exposure to48h was statistically significant (P<0.01). Size-dependent effect:exposed to48h, compared with the alumina nanoparticle (13nm) group and aluminium oxide-micro (10μm) group, the difference between the alumina nanoparticle (50nm) group was statistically significant (P<0.05).3. Mice bone marrow micronucleus testIn the female and male group, micronucleus of different doses were less than2%o.4. Mice sperm abnormality test(1) Normal mice sperm abnormality rate was between0.8%to3.4%, the rates of blank control group and low-, medium-dose groups of each size group were in normal range, the high-dose group was beyond the normal range and compared with the control group, the difference was statistically significant (P<0.01).The difference was not statistically significant (P>0.05) in the high-dose groups of each size group. (2) Mice testicular pathological results The number of spermatogenic cells of the mouse testis in the control group and low dose group were many, the levels of cells were clarity. The number of spermatogenic cells of the mouse testis and the level of cells in the medium-dose group were less than of the control group. The number of spermatogenic cells of the mouse testis and the level of cells in the high-dose group were lest than of the control group, the level disorganized, and spermatogenic cells formed irregular gaps.(3) Mice testicular oxidative stress The results of GSH-PX analyzed showed: dose-dependent effect:compared with the control group, with the increasing exposure dose, in the GSH-PX of different size groups there was a decreasing trend, the high significantly, and the difference with the medium-and high-dose was statistically significant (P<0.05, P<0.01). Size-dependent effect:the GSH-PX difference between different size groups was not statistically significant (P>0.05).The results of SOD analyzed showed that:dose-dependent effect:ompared with the control group, with the increasing exposure dose, in the SOD of different size groups there was a decreasing trend, the high significantly, and the difference with the high-dose of the alumina nanoparticle (13nm) and the aluminium oxide-micro (10μm) group was statistically significant (P<0.01), the difference with the medium-and high-dose of the alumina nanoparticle (50nm) was statistically significant (P<0.01). Size-dependent effect:the SOD difference between different size groups was not statistically significant (P>0.05).The results of MDA analyzed showed that:dose-dependent effect:compared with the control group, with the increasing exposure dose, in the MDA of different size groups there was an increasing trend, the high significantly, the MDA difference between different size groups was statistically significant (P<0.05). Size-dependent effect:the MDA difference between different size groups was statistically significant (P<0.05).The results of T-AOC analyzed showed that dose-dependent effect:compared with the control group, with the increasing exposure dose, in the T-AOC of different size groups there was a decreasing trend, the high significantly, and the difference with the high dose of the different size groups was statistically significant (P<0.01). Size-dependent effect:the T-AOC difference between differen tsize groups was not statistically significant (P>0.05).[Conclusion]1. Alumina nanoparticles (13nm and50nm) did not display any mutagenicity or affect the levels of oxidative stress-response systems.2. Alumina nanoparticles (13nm and50nm) can enter the cells and cause DNA damage with a time-and dose-and size-dependent manner, but compared with the positive control group, the DNA damage induced by alumina nanoparticles was weak; cause cellular oxidative stress response, we speculated that oxidative stress is one of the causes of DNA damage.3. The bone marrow micronucleus rates induced by alumina nanoparticles (13μm and50nm) were in the normal range. Mouse bone marrow micronucleus test was negative.4. Alumina nanoparticles (13nm and50nm) can damage the mouse testis structural, elevate sperm deformity rate, but compared with the positive control group, the sperm deformity rates by alumina nanoparticles were very low. Alumina nanoparticles (13nm and50nm) can induce oxidative stress in the dose-and size-dependent manner, we speculated that the sperm deformity rate and pathological damage by alumina nanoparticles were relevant with oxidative stress by alumina nanoparticles.5. According to the above, we suggested that alumina nanoparticles (13nm and50nm) may cause genotoxicity and that oxidative stress was involved in this process.6.This topic shortcomings:(1) Methodological limitations, although the standard toxicology tests were chosen in the topic, it needs a further check that if new or improved test methods will fit to the nanoparticles, as a new compound.(2) Though alumina nanoparticles own small size effect and surface and interface effect etc, its physical and chemical properties is nuclear.(3) Great difficult exists in how to extrapolate the results of tests and reflect reasonably the potential hazard to human induced by alumina nanoparticles. |
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