Objectives We investigated the genotoxic effects of 40-59 nm silver nanoparticles (Ag-NPs) by bacterial reverse mutation assay (Ames test), in vitro comet assay and micronucleus (MN) assay. In particular, we directly compared the effect of cytochalasin B (cytoB) and rat liver homogenate (S9 mix) in ...
Objectives We investigated the genotoxic effects of 40-59 nm silver nanoparticles (Ag-NPs) by bacterial reverse mutation assay (Ames test), in vitro comet assay and micronucleus (MN) assay. In particular, we directly compared the effect of cytochalasin B (cytoB) and rat liver homogenate (S9 mix) in the formation of MN by Ag-NPs. Methods Before testing, we confirmed that Ag-NPs were completely dispersed in the experimental medium by sonication (three times in 1 minute) and filtration ($0.2{\mu}m$ pore size filter), and then we measured their size in a zeta potential analyzer. After that the genotoxicity were measured and especially, S9 mix and with and without cytoB were compared one another in MN assay. Results Ames test using Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains revealed that Ag-NPs with or without S9 mix did not display a mutagenic effect. The genotoxicity of Ag-NPs was also evaluated in a mammalian cell system using Chinese hamster ovary cells. The results revealed that Ag-NPs stimulated DNA breakage and MN formation with or without S9 mix in a dose-dependent manner (from $0.01{\mu}g/mL$ to $10{\mu}g/mL$). In particular, MN induction was affected by cytoB. Conclusions All of our findings, with the exception of the Ames test results, indicate that Ag-NPs show genotoxic effects in mammalian cell system. In addition, present study suggests the potential error due to use of cytoB in genotoxic test of nanoparticles.
Objectives We investigated the genotoxic effects of 40-59 nm silver nanoparticles (Ag-NPs) by bacterial reverse mutation assay (Ames test), in vitro comet assay and micronucleus (MN) assay. In particular, we directly compared the effect of cytochalasin B (cytoB) and rat liver homogenate (S9 mix) in the formation of MN by Ag-NPs. Methods Before testing, we confirmed that Ag-NPs were completely dispersed in the experimental medium by sonication (three times in 1 minute) and filtration ($0.2{\mu}m$ pore size filter), and then we measured their size in a zeta potential analyzer. After that the genotoxicity were measured and especially, S9 mix and with and without cytoB were compared one another in MN assay. Results Ames test using Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains revealed that Ag-NPs with or without S9 mix did not display a mutagenic effect. The genotoxicity of Ag-NPs was also evaluated in a mammalian cell system using Chinese hamster ovary cells. The results revealed that Ag-NPs stimulated DNA breakage and MN formation with or without S9 mix in a dose-dependent manner (from $0.01{\mu}g/mL$ to $10{\mu}g/mL$). In particular, MN induction was affected by cytoB. Conclusions All of our findings, with the exception of the Ames test results, indicate that Ag-NPs show genotoxic effects in mammalian cell system. In addition, present study suggests the potential error due to use of cytoB in genotoxic test of nanoparticles.
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제안 방법
The Olive Tail Moment (OTM; tail distance × percentage of DNA in tail) was used to quantify DNA damage, based on the random scoring of 100 nuclei per slide.
Among various kinds of nanoparticles, silver nanoparticles (Ag-NPs) are the most commercialized nanoparticles according to the Woodrow-Wilson database which is a data source for information on products based on nanotechnology. The purpose of this study was to investigate the potential genotoxic effects of Ag-NPs using the Ames test in four different bacterial strains and the in vitro comet and MN assays in Chinese hamster ovary (CHO-K1) cells. In particular, we directly compared the effect of cytoB and S9 mix in the formation of MN by Ag-NPs.
The plates were then incubated at 37˚C for 48 hours, after which revertants and surviving colonies were counted. Three independent experiments were conducted and each experiment consisted of three replicate plates for each treatment. The positive control used in the absence of S9 mix was 2-nitrofluorene for the TA98 strain, sodium azide for the TA100 and TA1535 strains, and 9-aminoacridine hydrochloride for the TA1537 strain.
To identify chromosomal damage in CHO-K1 cells exposed to Ag-NPs, we carried out CBMN and MN assays (Figure 3). The CBMN assay was carried out in the presence or absence of S9 mix.
대상 데이터
CHO-K1 cells were obtained from the Korean Cell Line Bank (Seoul, Korea). CHO-K1 cells were grown in RPMI1640 supplemented with 5% fetal bovine serum, penicillin (100 units/ mL) and streptomycin (100 μg/mL) at 37˚C in an atmosphere of 5% CO 2/95% air under saturated humidity.
데이터처리
Differences between groups were determined by Duncan’s post hoc test following one-way analysis of variance.
성능/효과
In conclusion, Ag-NPs did not induce mutations in the Ames test. However, Ag-NPs exhibited dose-dependent genotoxicity in the comet and MN assays in CHO-K1 cells.
The results of the CBMN and MN assays without S9 mix were significantly different (p < 0.05).
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