| ObjectiveNanomaterials are engineered materials approximately1to100nanometers in atleast one dimension. Being smaller than100nm in diameter, nanomaterials have anextended surface compared to the bulk forms with regard to their mass. Thus,nanomaterials exhibit specific physico-chemical properties and functions. The smallsize in addition to the novel physico-chemical properties may bring new challenges tohuman health and environmental safety. Therefore, it is one of the biggest challengesfor us whether the current toxicological testing methods are suitable for the riskevaluation and safety assessment of the nanomaterials. It is very imperative to solvethe problem in nanotoxicological studies.A key area governing health risk assessment of new chemicals is genotoxicology,involving the study of genetic damage following exposure to test substances. Suchinformation is vital as DNA damage could not only initiate cancer development, butalso have an impact upon fertility and the health of subsequent generations ifdisturbances arise in reproductive cells. Due to their small size and high surface area,coupled to other physico-chemical features such as metal contaminants and chargedsurfaces, nanomaterials may have unpredictable genotoxic properties, while themechanism of genotoxicity may be different from the bulk materials. Thus, it’scriticized whether the nanomaterials can be evaluated the characteristics ofgenotoxicity using the current genotoxic testing methods. Owing to its complexity andimportance, a new scientific research discipline---nanogenetoxicology emerges.The data accumulating in the literature does point to some nanomaterialsharbouring the ability to damage DNA, but given the inconsistencies it is difficult todraw conclusions. There was low consistence between the Comet assay, micronucleusassay and Ames tests, which might indicate the specific character of genotoxicity fornanomaterials or Ames test unsuitable for detecting genotoxicity induced bynanomaterials. In order to solve the significant problem, it is therefore crucial that thegenotoxic potential of nanomaterials and underlying mechanisms of action are clarified.Furthermore, there is a distinct need to identify associations between genotoxicresponses of nanomaterials and its specific physico-chemical features. Nanomaterialsmay have unpredictable genotoxic properties. They may cause DNA damage indirectly,by promoting oxidative stress and inflammatory responses. Alternatively, if small enough, they may pass through cellular membranes and gain access to the nucleuswhere they may interact directly with DNA, causing damage. Additionally, ifnanomaterials were able to accumulate within a cell but not necessarily gain access tothe nucleus, they may still come into direct contact with DNA during mitosis when thenuclear membrane breaks down, providing ample opportunity for DNA aberrations toarise. The known genotoxic endpoints cover DNA damage, gene mutation,chromosome aberration and changes of hereditary substances integrity. Therefore, inorder to reveal the genotoxic potential of nanomaterials and underlying mechanisms ofaction, genotoxic assays should cover all the underlying mechanisms of action fornanomaterials, while the known genotoxic endpoints should be included. Thus, one canselect a battery of genotoxicity tests including many endpoints, but there exists a mainshortcoming for combined assays because the results analysis is very difficult forinconsistent doses and effects of different experimental tests. Alternatively, we providethe cytokinesis—block micronucleus cytome assays （CBMN Cyt assay）, which is acomprehensive method for measuring chromosome breakage, DNA misrepair,chromosome loss, non-disjunction, necrosis, apoptosis and cytostasis from manygenotoxic endpoints. The CBMN Cyt assay basically covers the possible mechanismsof action for nanomaterials; while a variety of genetic toxicology endpoints areincluded. Meanwhile, it avoids the main shortcoming of combined assays.Our research is aimed to conduct comparative analysis of characteristics ofgenotoxicity for the identical composition bulk cadmium sulfide(bulk CdS) andnanoscale cadmium sulfide(nanoscale CdS), five typical dendrimers and joint effect ofnanoscale CdS and Mitomycin C(MMC), nanoscale zinc oxide(nanoscale ZnO)conducted using CBMN Cyt assay which cover the potential mechanisms of actions fornanomaterials and can be applied to investigate the characteristics of genotoxicity froma variety of genetic endpoints. Furthermore, characteristics of genotoxicity ofrepresentative nanomaterials are thoroughly investigated using the established method,meanwhile the relationship between the physio-chemical features of nanomaterial andits genotoxicity are exploited to reveal the potential characteristics of genotoxicity.Therefore, these studies will provide a scientific basis for the construction ofgenotoxicity test system for nanomaterials. MethodsMouse lymphoma cell L5178Y was obtained from the type culture collection ofChina Academy of Science (Shanghai, China) with cell strain determined.Cells were treated with various concentrations of nanoscale CdS and5typical dendrimers(DAB-Am-4，polypropylenimine tetramine dendrimer, generation1；PAMAM dendrimer，ethylenediamine core, generation5.0solution；polyester bis-MPA dendron，16carboxyl,1amine；hyperbranched bis-MPA polyester-64-hydroxyl，generation4and poly(ethylene glycol)，16acetylene dendron, generation3.During the logarithmic phase of cultivated L5178Y cells, they were seeded into24well cell culture clusters at the concentration of4×105cells/ml and treated withvarious concentrations of nanoscale CdS and5typical dendrimers for4hours.50μlculture medium was added in the negative control group and50μl MMC was added inthe positive control group to make up final concentration of0.1μg/ml.Set up duplicatecultures per group and or treatment studied. Then,20μl Cyto-B was added to make upfinal concentration of4.5μg/ml and returned the well cell culture to incubator and thenincubated the culture for additional24hours. After cultivating for28hours, the cellswere harvested and treated twice with0.075M of potassium chloride hypotonicsolution for7minutes and fixation solution (a mixture of methanol and acetic acid;3:1). Air-dried cell preparations were stained with10%Giemsa solution for15minutes.Treatment dose was set based on the IC50value of the test nanomaterial, whichranged from the highest dose group (1/3~1/2IC50) to the lowest dose group (1/48~1/32IC50). At least5slides,2000binucleated cells per slide and a total of10,000binucleated cells per treatment group were scored for MNi, NPBs and NBUDsaccording to cytokinesis-block micronucleus cytome assay protocol by Fenech.Nanomaterials:LumidotTM CdS-6， quantum dot nanoparticles kit was purchased fromSigma-Aldrich. The Kit contains six toluene solutions of CdS quantum dotnanocrystals with fluorescence emission maximum spanning UV-blue spectrum. Thetest Lumidot CdS380nanocrystal is surface-stabilized with oleic acid coating,whose size is1.6nm.5typical dendrimers including DAB-Am-4， polypropylenimine tetraminedendrimer, generation1(molecular weight316.53g/mol, formula C16H40N6), PAMAMdendrimer； ethylenediamine core, generation5.0solution(molecular weight28824.81g/mol, formula C1262H2528N506O252C1262H2528N506O252)；polyester bis-MPA dendron，16carboxyl,1amine （bis-MPA dendron）； hyperbranched bis-MPApolyester-64-hydroxyl，generation4（ALH-64-OH）and poly(ethylene glycol)，16acetylene dendron, generation3were all purchased from Sigma-Aldrich.Results1. Comparison of genotoxicity between bulk CdS and nanoscale CdSThe mouse lymphoma cell L5178Y was exposed to0.0063，0.0125，0.025，0.05and0.1μg/ml of bulk or nanoscale CdS. The results indicated that the frequency oftotal micronucleus, type Ⅰ micronucleus and type Ⅱ micronucleus formation rosedramatically in bulk CdS group comparing with negative control at the concentrationof0.0125μg/ml. Dose dependent increase of total micronucleus, type Ⅰ micronucleus,type Ⅱ micronucleus, nucleoplasmic bridges and nulear buds were observed above0.0125μg/ml. However, comparing with negative control, the frequency of totalmicronucleus, type Ⅰ micronucleus, type Ⅱ micronucleus and nucleoplasmic bridgesplummet significantly in nanoscale CdS group at the concentration of0.0063μg/ml,while the frequency of nuclear bud increased at the concentration of0.0125μg/ml andthen the frequency of total micronucleus also rose at0.025μg/ml. All indexes includingtotal micronucleus, type Ⅰ micronucleus, type Ⅱ micronucleus, nucleoplasmicbridges and nulear buds elevated dramatically above or equal to0.05μg/ml.Comparing the same concentration of bulk CdS with nanoscale CdS, Theconclusion revealed that the frequency of type Ⅱ micronucleus and nucleoplasmicbridges in bulk CdS groups were significantly higher than those in nanoscale CdSgroups, and the nuclear buds in bulk CdS groups was dramatically lower than that innanoscale CdS.The time-effect analysis (9h,18h,27h and36h) results indicated that statisticallysignificant differences existed among these four time points on the indexes of totalmicronucleus, type Ⅰ micronucleus, type Ⅱ micronucleus, nucleoplasmic bridges andnuclear buds no matter bulk CdS or nanoscale CdS. The frequencies of totalmicronucleus, type Ⅰ micronucleus, type Ⅱ micronucleus, nucleoplasmic bridges andnuclear buds reached peak during18h to27h.2. Genotoxicity of five typical dendrimers（1） DAB-Am-4, polypropylenimine tetramine dendrimer, generation1 Under the final concentration of0.5，1，2，4，8μg/ml of DAB-Am-4,polypropylenimine tetramine dendrimer, generation1，DAB-Am-4, polypropyleniminetetramine dendrimer, generation1could increase the formation of nuclear buds at theconcentration of0.5μg/ml and the total micronucleus, type Ⅰ micronucleus, type Ⅱmicronucleus formation increased dose dependent manner above1μg/ml.The time-effect analysis results showed that statistically significant differenceswere observed among9h,18h,27h and36h of DAB-Am-4, polypropyleniminetetramine dendrimer, generation1incubation on the frequencies of the totalmicronucleus, type Ⅰ micronucleus, type Ⅱ micronucleus, nucleoplasmic bridges andnuclear buds. The indexes of total micronucleus, type Ⅰ micronucleus, type Ⅱmicronucleus, nucleoplasmic bridges and nuclear buds reached highest during18h to27h.（2） PAMAM dendrimer, ethylenediamine core, generation5.0The final concentration0.625，1.25，2.5，5，10μg/ml of PAMAM dendrimer,ethylenediamine core, generation5.0was given to the cells. The frequency of nuclearbuds increased at the concentration of1.25μg/ml and dose dependent increase on theformation of total micronucleus, type Ⅰ micronucleus, and type Ⅱ micronucleus aswell with nuceloplasmic bridges was oberserved above2.5μg/ml.The time-effect analysis results indicated that statistically significant differencesexisted among9h,18h,27h and36h four time points on the indexes of totalmicronucleus, type Ⅰ micronucleus, nucleoplasmic bridges and nuclear buds. Theindexes of total micronucleus, type Ⅰ micronucleus, nucleoplasmic bridges andnuclear buds reached highest during18h to27h. There was not significant differenceamong four time points on the frequency of type Ⅱ micronucleus.（3） Polyester bis-MPAdendron,16carboxyl,1amineThe cells were exposed to25，50，100，200，400and800μg/ml of polyesterbis-MPA dendron,16carboxyl,1amine, polyester bis-MPA dendron,16carboxyl,1amine could increase the formation of nuclear buds and type Ⅰ micronucleus at theconcentration of50μg/ml and dose dependent increase of total micronucleus, type Ⅰmicronucleus, type Ⅱ micronucleus and nuceloplasmic bridges was observed above100μg/ml.The time-effect analysis (9h,18h,27h and36h) results showed that there wasstatistically significant difference among four time points on the indexes of total micronucleus, type Ⅰ micronucleus, type Ⅱ micronucleus, nucleoplasmic bridges andnuclear buds.（4） Hyperbranched bis-MPApolyester-64-hydroxyl, generation4Under the final concentration of62.5，125，250，500and1000μg/mlhyperbranched bis-MPA polyester-64-hydroxyl, generation4, hyperbranched bis-MPApolyester-64-hydroxyl, generation4increased the frequency of nucleoplasmic bridgesat the concentration of125μg/ml and the total micronucleus, type Ⅰ micronucleus,type Ⅱ micronucleus and nuclear buds rose in dose dependent manner above250μg/ml.The time-effect analysis (9h,18h,27h and36h) results indicated that statisticallysignificant differences existed among four time points on the frequency of totalmicronucleus, type Ⅰ micronucleus, type Ⅱ micronucleus, nucleoplasmic bridges andnuclear buds. The frequency of total micronucleus, type Ⅰ micronucleus, type Ⅱmicronucleus, nucleoplasmic bridges and nuclear buds were highest during18h to27h.（5） Poly(ethylene glycol)，16acetylene dendron, generation3Under the final concentration0.625，1.25，2.5，5and10mg/ml of poly(ethyleneglycol)，16acetylene dendron, generation3，poly(ethylene glycol)，16acetylenedendron, generation3increased the total micronucleus and type Ⅰ micronucleusfirstly at the concentration of2.5mg/ml and the total micronucleus, type Ⅰmicronucleus, type Ⅱ micronucleus and nuclear buds rdse dose dependent at10mg/ml.3. Genotoxicity of joint effect of nanoscale CdS and MMC, nanoscale ZnOComparing with negative control, the frequency of total micronucleus, type Ⅰmicronucleus, type Ⅱ micronucleus and nucleoplasmic bridges plummet significantlyin nanoscale CdS group not only at the concentration of0.0063μg/ml but also at theconcentration of0.0031μg/ml, which indicates that nanoscale CdS can arouse hormesiseffect and suppress the DNA damage at lower dose. Genotoxicity results of joint effectof low dose nanoscale CdS and MMC, nanoscale ZnO show that nanoscale CdS caninhibit the formation of total micronucleus, type I micronucleus, type II micronucleus,nucleoplasmic bridges and nuclear buds, while dose dependent decrease of totalmicronucleus, type Ⅰ micronucleus, type Ⅱ micronucleus, nucleoplasmic bridges andnulear buds were observed.Compared with cells treated with positive mutagen MMC(0.1μg/ml), the frequency of total micronucleus, type Ⅰ micronucleus, type Ⅱ micronucleus,nuceloplasmic bridges and nuclear buds decreased significantly in nanoscaleCdS(0.0063μg/ml) in liaison with MMC(0.1μg/ml) group. While the frequency of totalmicronucleus, type Ⅰ micronucleus, type Ⅱ micronucleus, nuceloplasmic bridges andnuclear buds plummeted even dramatically in the group of0.0031μg/ml nanoscale CdSin liaison with0.1μg/ml MMC. There was no significant difference on the frequency oftype Ⅱ micronucleus between0.0031μg/ml nanoscale CdS in liaison with0.1μg/mlMMC group and negative control.Compared with cells treated with positive mutagen MMC(0.1μg/ml), thefrequency of total micronucleus, type Ⅰ micronucleus, type Ⅱ micronucleus,nuceloplasmic bridges and nuclear buds decreased dramatically in nanoscaleCdS(0.0063μg/ml) in liaison with MMC(0.05μg/ml) group. There was no significantdifference on the frequency of nuceloplasmic bridges between0.0063μg/ml nanoscaleCdS in liaison with0.05μg/ml MMC group and negative control. While all theseindexes plummeted even significantly in the group of0.0031μg/ml nanoscale CdS inliaison with0.05μg/ml MMC. The frequency of nuceloplasmic bridges and nuclearbuds didn’t differ significantly between0.0031μg/ml nanoscale CdS in liaison with0.05μg/ml MMC group and negative control.Compared with the final concentration of5μg/ml nanoscale ZnO group, thefrequency of total micronucleus, type Ⅰ micronucleus, type Ⅱ micronucleus,nuceloplasmic bridges and nuclear buds in nanoscale CdS(0.0063μg/ml) in liaison withnanoscale ZnO(5μg/ml) group decreased greatly, while the frequency of totalmicronucleus, type Ⅰ micronucleus and nuclear buds in nanoscale CdS(0.0031μg/ml)in liaison with nanoscale ZnO(5μg/ml) group decreased even significantly, there wasno significant difference on the type Ⅱ micronucleus between nanoscaleCdS(0.0031μg/ml) in liaison with nanoscale ZnO(5μg/ml) group and negative control.Compared with the final concentration of2.5μg/ml nanoscale ZnO group, thefrequency of total micronucleus, type Ⅰ micronucleus, type Ⅱ micronucleus,nuceloplasmic bridges and nuclear buds plummeted dramatically in the group of0.0063μg/ml nanoscale CdS in liaison with2.5μg/ml nanoscale ZnO and there were nostatistically significant differences on the frequency of total micronucleus, type Ⅱmicronucleus, nuceloplasmic bridges and nuclear buds between this nanoscaleCdS(0.0063μg/ml) in liaison with nanoscale ZnO(2.5μg/ml) group and negativecontrol. While the frequency of total micronucleus, type Ⅰ micronucleus, type Ⅱ micronucleus, nuceloplasmic bridges and nuclear buds plummeted even fiercely in thegroup of0.0031μg/ml nanoscale CdS in liaison with2.5μg/ml nanoscale ZnO. Onlythe type Ⅰ micronucleus in0.0031μg/ml nanoscale CdS in liaison with2.5μg/mlnanoscale ZnO group was a little higher than that in the negative control.ConclusionsThere exists significant difference on the genotoxic effect and genotoxiccharacteristics between bulk CdS and nanoscale CdS groups. The bulk CdS can inducethe DNA damage directly, while nanoscale CdS can modulate the homestasis of DNAbidirectionally, which arouses hormesis effect, suppress the DNA damage at lowerdose and induces the DNA damage at higher dose. The frequency of nuclear budskyrockets firstly in lower dose of nanoscale CdS and then the else indexes, includingthe total micronucleus, type I micronucleus, type II micronucleus and nucleoplasmicbridges rise with the increasement of dose, whilst the frequency of type I micronucleusand type II micronucleus rise in low dose of bulk CdS groups. The DNA damage inbulk CdS group is even worse than that in nanoscale CdS group in the range of lowdose.All of the five typical dendrimers can induce DNA damage directly. As to thesame genotoxic effect, the given dose is sequenced as the following: poly(ethyleneglycol)，16acetylene dendron, generation3> hyperbranched bis-MPApolyester-64-hydroxyl，generation4> polyester bis-MPA dendron，16carboxyl,1amine>> PAMAM dendrimer， ethylenediamine core, generation5.0solution>DAB-Am-4，polypropylenimine tetramine dendrimer, generation1.The frequency of nuclear bud rises firstly in low dose of DAB-Am-4，polypropylenimine tetramine dendrimer, generation1and PAMAM dendrimer，ethylenediamine core, generation5.0solution group and then the else indexes emerges,which reach the peak during18h to27h. The frequency of type I micronucleus andnuclear bud rise firstly in low dose of polyester bis-MPA dendron，16carboxyl,1aminegroup. hyperbranched bis-MPA polyester-64-hydroxyl，generation4can arouse theincreased frequency on the nucleoplasmic bridges firstly and then the else indexesreach highest during18h to27h. The frequency of type I micronucleus rises greatly inlow dose of poly(ethylene glycol)，16acetylene dendron, generation3. The genotoxiceffect is correlated well with its central core and functional groups on exterior surfaceof these dendrimers.Genotoxicity results of joint effect of nanoscale CdS and MMC, nanoscale ZnO indicate that nanoscale CdS at low dose can induce the hormesis effect and inhibit thegenotoxicity of MMC and nanoscale ZnO on the formation of total micronucleus, typeI micronucleus, type II micronucleus, nucleoplasmic bridges and nuclear buds, thusnanoscale CdS can suppress the frequency of total micronucleus, type I micronucleus,type II micronucleus, nucleoplasmic bridges and nuclear buds at low dose. |
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