DOI 10.1007/s10517-015-2868-y
812
Bulletin of Experimental Biology and Medicine, Vol. 158, No. 6, April, 2015 NANOTECHNOLOGIES
Genotoxicity of Single-Walled Carbon Nanotubes: In Vitro Study on Human Embryonic Fibroblast Cells V. A. Nikitina1, A. I. Chausheva 1 , I. A. Suetina 2 , L. D. Katosova 1, D. G. Zheglo 1, M. V. Mezentseva 2 , V. I. Platonova 1, Yu. A. Revazova3, and S. I. Kutsev 1,4 Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 158, No. 12, pp. 783-786, December, 2014 Original article submitted July 1, 2014 The effects of single-walled carbon nanotubes on the levels of DNA aberrations, chromosome and genome disorders were studied on human embryonic fibroblasts, their karyotype was analyzed by the spectral karyotyping method. The level of DNA aberrations increased after 3-h exposure to the nanotubes. No appreciable increase in the incidence of aberrant metaphases, micronuclei, and chromosome 1, 6, 8, 11, X, and Y aneuploidy after 24- and 48-h incubation with the nanotubes were detected. Key Words: single-walled carbon nanotubes; human embryonic fibroblasts; spectral karyotyping; DNA aberrations; fluorescent in situ hybridization The use of nanoparticles (NP) is a promising technology for the manufacturing cosmetic articles, drugs, biomimetics, lubricants, and fuel additives, protective and fortifying films, composite, textile, packing materials, lacquers and paints [3]. The creation of nanoindustry is a priority trend of economy in Russia; carbon NP is one of the priorities in the development of materials [1,6]. The possibility of using carbon NP in biotechnology and medicine as biosensors, means for addressed drug delivery, in creation of 3D tissue engineering constructions is demonstrated [10,14]. Sublayers of carbon NP are effective supporting matrix; moreover, they stimulate proliferative activity of cells [7] that can be useful in cell therapy and tissue engineering. Hence, a wide spectrum of technologies and applications of nanomaterials leads to their higher production and their inevitable release into the environment and to contacts with humans. Physicochemical characteristics of NP may differ significantly 1 Medical Genetic Research Center, Russian Academy of Medical Sciences; 2D. I. Ivanovsky Research Institute of Virology, N. F. Gamaleya Federal Research Institute of Epidemiology and Microbiology, Moscow; 3F. F. Erisman Federal Research Center of Hygiene, Mytishchi, Moscow Region; 4N. I. Pirogov Russian National Research Medical University, Ministry of Health of the Russian Federation, Moscow, Russia. Address for correspondence: [email protected]. V. A. Nikitina
from the materials of an equivalent composition, but of larger size, and hence, the data on their cyto- and genotoxicity cannot be directly extrapolated to NP. We studied the genotoxicity of single-walled carbon nanotubes (SWCNT) on human embryonic fibroblasts (HEF) by the methods fit to characterize the possible clastogenic and aneugenic effects of the nanotubes.
MATERIALS AND METHODS DNA breaks emerging within the first 3 h after addition of SWCNT, were evaluated by the DNA comet method. Cytogenetic study of the level of chromosome aberrations was carried out after 24 h, when most cells started division (mitosis metaphase). The aneugenic characteristics of SWCNT were evaluated by analyzing the incidence of micronuclei and aneuploidies by chromosomes 1, 6, 8, 11, X, and Y after the first cell division of HEF after 48 h. Characteristics of SWCNT. SWCNT paste (1 nm in diameter for a solitary nanotube and 10-30 nm in diameter for a bundle of nanotubes) was a kind gift from A. V. Krestinin (Institute of Chemical Physics Problems, Russian Academy of Sciences, Chernogolovka). The paste was autoclaved (1 atm, 120oC,
0007-4888/15/15860812 © 2015 Springer Science+Business Media New York
V. A. Nikitina, A. I. Chausheva, et al.
813
30 min), after which 1% SWCNT in 0.9% NaCl was prepared. Subtoxic concentration of SWCNT corresponding to 1/1000 dilution was selected on the base of previous experiments [2] with the use of MTT test. Characteristics and culturing of HEF cells. The HEF cells were inoculated in a concentration of 100,000 cells/ml and cultured in Eagle’s medium with 10% fetal calf serum (HyClone). The SWCNT preparations were incubated during 3, 24, or 48 h. DNA comet assay. After incubation with SWCNT, the cells were scraped from the bottom of the flask with a silicon spatula, transferred into tubes, and centrifuged. The resultant precipitate was suspended in buffer to the final concentration of 1.5×105 cells/ml. Study by the DNA comet assay (alkaline version) was carried out as described previously [5]. Micropreparations were stained with SYBR Green I fluorescent dye. The DNA comets were analyzed using CASP 1.2.2 software. The percent content of DNA in the comet tail (tail DNA%) served as the indicator of DNA aberrations. Cells with different levels of DNA aberrations were distributed into 5 groups: 0.5, 5.1-10, 10.1-15, 15.1-20 and more tail DNA%. Method for registration of chromosome aberrations. After colchicine treatment, the cells were washed from culture medium and removed from the bottom of the flask with Trypsin–EDTA. The resultant cell suspension was incubated with hypotonic solution (0.05% KCl, 10 min, 37oC) and fixed 3 times (methanol:glacial acetic acid, 3:1), after which the cytogenetic preparations were made by the standard dry air method. The preparations were stained with azure and eosin. Cells with solitary, paired fragments, chromosome and chromatid exchanges were counted. Spectral analysis of chromosomes. Spectral karyotyping (SKY), denaturing, and hybridization of probes and metaphase chromosome preparations
was carried out simultaneously in a hybridizer (ThermoBrite; StatSpin Inc.). A set of specific probes for spectral analysis of chromosomes (Applied Spectral Imaging Inc.) was used. Washing and labeling with antibodies were carried out according to the standard protocol, the images were analyzed by HiSKY 5.5 software (Applied Spectral Imaging Inc.). Micronuclear test. The cells were removed from the flask with Trypsin–EDTA solution, incubated in hypotonic solution (0.55% KCl, 5 min, 4oC), and fixed 3 times (methanol:glacial acetic acid, 3:1). Cell suspensions were applied onto dry cold slides, put on a coolant for 5 min, and dried in the air. The preparations were stained with Acridine Orange, then with DAPI. Interphase FISH analysis. Centromere-specific DNA probes for chromosomes X (DXZ1), Y (DYZ3), 1 (D1Z5), 6 (D6Z1), 8 (D8Z1), and 11 (D11Z1) (Vysis; Abbott) were used. Denaturing, hybridization, and washing were carried out by the standard method. Hybridization mixture for two-color FISH contained samples for three chromosome pairs: X and Y, 1 and 8, 6 and 11. Analysis was carried out as described previously [3], a total of 500 interphase nuclei were analyzed for each chromosome pair three times. Cells with one or three signals by one chromosome and two signals by the other (for pairs 1 and 8, 6 and 11), without signals or with two signals by one chromosome and one signal by the other chromosome (for X and Y pair) were referred to aneuploid. The results were analyzed by Statistica software. Comparative study was carried out by Student’s t and Pearson’s χ2 tests.
RESULTS The HEF cell karyotype was studied by the SKY method before experiment. Twenty-five metaphase
Fig. 1. Spectral analysis of HEF cell karyotype. a) Karyotype (46,XY); b) derivative chromosome t(4:17)(17qter→17p11::4q21→4qter).
Bulletin of Experimental Biology and Medicine, Vol. 158, No. 6, April, 2015 NANOTECHNOLOGIES
814
TABLE 1. Percentage of Cells with Different Levels of DNA Aberrations after Incubation with SWCNT Control (n=239)
SWCNT (n=292)
Parameter abs. Tail DNA %
%
abs.
%
0-5
165
69
192
66
5-10
40
17
47
16
10-15
20
8
22
7.5
15-20
7
3
4
1.5
>20
7
3
27
9
Note. Percent of total number of analyzed cells is shown. p=0.039, df=4, χ2=10.094.
plates were analyzed. The karyotype of 23 plates corresponded to normal male karyotype – 46,XY (Fig. 1, a), while 2 cells contained a derivative chromosome formed by translocation of chromosomes 4 and 17 (Fig. 1, b). The mean level of DNA aberrations in control was 4.58±0.40% (median 2.13); it increased (p=0.04) after 3-h exposure with SWCNT (6.10±0.58 tail DNA%, median 1.97). Analysis of distribution of cells with different levels of DNA aberrations (Table 1) showed that the mean level of aberrations increased after exposure with SWCNT at the expense of higher level of severely damaged cells (more than 20% of tail DNA) vs. control. Comparison of distributions in the control and experiment (df=4, χ2=10.094, p=0.039) confirmed this result. Structural and quantitative mutations were evaluated by chromosomal analysis methods. The incidence of chromosome breaks and translocations was evalua-
ted by analysis of the metaphase plate chromosomes. The level of spontaneous chromosome aberrations was 4.50±0.04% – virtually the same as after incubation with SWCNT – 5.40±0.44% (Table 2). Comparison of the distributions of damaged and intact metaphases in the control and after exposure with SWCNT showed no appreciable differences (df=1, χ2=1.549, p=0.213). The spectrum of chromosome aberrations included mainly chromatid, rarely chromosome fragments and exchanges (Table 2). The incidence of micronuclei, characterizing aneugenic (chromosome lag in the mitosis anaphase) and clastogenic (chromosome break) qualities, did not change after incubation with SWCNT (df=1, χ2=2.21, p=0.14; Fig. 2). The incidence of chromosomes 1, 6, 8, 11, X, and Y aneuploidy was virtually the same in control cultures and in cultures incubated with SWCNT according to analysis with the use of fluorescent centromere samples in interphase nuclei of HEF cells (Table 3). A higher incidence of chromosome Y aneuploidy in comparison with other chromosomes was detected. High incidence of spontaneous DNA and chromosome aberrations in HEF cells is worthy of note; the level of chromosome aberrations in the cells was 1.5-2.0 times higher than in healthy volunteers’ whole blood lymphocytes (2-3% of aberrant metaphases, according to database [4]). No special studies of tissue culture genetic stability have been presented. It seems that a higher changeability in comparison with short-lived lymphocyte culture, traditionally used for evaluation of spontaneous mutagenesis in humans, is characteristic of cultured cells. Hence, the level of DNA aberrations increased in HEF cells incubated with SWCNT, though the incidence of chromosome aberrations, micronuclei, and aneuploidy changed just negligibly. Our results
TABLE 2. Levels of Chromosome Aberrations in Cells Incubated with SWCNT Parameter
Control
SWCNT
950
950
43 (4.5%)
55 (5.44%)
solitary fragments
2.66
3.44
paired fragments
1.78
1.88
chromosome exchanges
0.13
0.12
0
0.18
Total number of metaphases Number of aberrant metaphases Number of structural aberrations/100 cells
chromatid exchanges
Note. *Percent of total level is shown in parentheses. χ2=1.549.
Fig. 2. Incidence of micronuclei in HEF cells. No significant differences detected. df=1, χ2=2.21, p=0.14.
V. A. Nikitina, A. I. Chausheva, et al.
815
TABLE 3. Incidence of Aneuploid Nuclei after SWCNT Exposure Control
SWCNT χ2
Chromosome diploid
aneuploid
diploid
aneuploid
X
980
20
978
22
0.1 (р=0.75)
Y*
955
45
937
63
3.17 (р=0.07)
1
975
25
981
19
0.84 (р=0.36)
8
987
13
985
15
0.14 (р=0.7)
6
983
17
985
15
0.13 (р=0.72)
11
982
18
980
20
0.11 (р=0.74)
Note. *Significant differences in distribution of incidence of chromosome Y aneuploidy in comparison with X, 1, 6, 8, and 11 in control cells and cells exposed to SWCNT.
just partially confirm the data on SWCNT genotoxicity, indicating a higher level of DNA aberrations, micronuclei, and aneuploidy [8,9,11,12]. Induction of ROS, accumulation of LPO products and DNA aberrations (single, double-strand breaks, modified bases, DNA sutures between strands) in the cells and apoptosis triggering are traditionally assumed to be the main mechanisms of the cyto- and genotoxic activities of NP [13]. The possible effects of nanotubes on chromosome deviations in mitosis and on the DNA molecule conformation status are demonstrated [12]. The potential hazards of mutagenic and carcinogenic effects of nanomaterials intended for prospective medical use necessitate further research in genonanotoxicology.
REFERENCES 1. M. V. Alfimov, Ross. Nanotekhnol., 4, Nos. 1-2, 1-8 (2009). 2. I. I. Bobrinetskii, A. S. Seleznyov, I. A. Gaiduchenko, et al., Biofizika, 58, No. 3, 524-530 (2013).
3. N. P. Bochkov, V. A. Nikitina, E. S. Voronina, and N. P. Kuleshov, Bull. Exp. Biol. Med., 148, No. 4, 677-683 (2010). 4. N. P. Bochkov, A. N. Chebotaryov, L. D. Katosova, and V. I. Platonova, Genetika, 37, No. 4, 549-557 (2001). 5. A. K. Zhanataev, V. A. Nikitina, E. S. Voronina, and A. D. Durnev, Prikladn. Toksikol., 2, No. 4, 27-37 (2011). 6. A. V. Krestinin, Ross. Nanotekhnol., 2, Nos. 5-6, 18-23 (2007). 7. R. Ya. Podchernyaeva, I. A. Suetina, G. R. Mikhailova, et al., Vopr. Virusol., 57, No. 5, 46-48 (2012). 8. J. Cveticanin, G. Joksic, A. Leskovac, et al., Nanotechnology, 21, No. 1, doi: 10.1088/0957-4484/21/1/015102 (2010). 9. N. R. Jacobsen, G. Pojana, P. White, et al., Environ. Mol. Mutagen., 49, No. 6, 476-487 (2008). 10. Y. Lin, S. Taylor, H. Li, et al., J. Mater. Chem., No. 14, 527541 (2004). 11. L. Migliore, D. Saracino, A. Bonelli, et al., Environ. Mol. Mutagen., 51, No. 4, 294-303 (2010). 12. L. M. Sargent, A. A. Shvedova, A. F. Hubbs, et al., Environ. Mol. Mutagen., 50, No. 8, 708-717 (2009). 13. N. Singh, B. Manshian, G. J. Jenkins, et al., Biomaterials, 30, Nos. 23-24, 3891-3941 (2009). 14. F. Valentini, M. Carbone, and G. Paleschi, Anal. Bioanal. Chem., 405, Nos. 2-3, 451-465 (2013).
812
Bulletin of Experimental Biology and Medicine, Vol. 158, No. 6, April, 2015 NANOTECHNOLOGIES
Genotoxicity of Single-Walled Carbon Nanotubes: In Vitro Study on Human Embryonic Fibroblast Cells V. A. Nikitina1, A. I. Chausheva 1 , I. A. Suetina 2 , L. D. Katosova 1, D. G. Zheglo 1, M. V. Mezentseva 2 , V. I. Platonova 1, Yu. A. Revazova3, and S. I. Kutsev 1,4 Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 158, No. 12, pp. 783-786, December, 2014 Original article submitted July 1, 2014 The effects of single-walled carbon nanotubes on the levels of DNA aberrations, chromosome and genome disorders were studied on human embryonic fibroblasts, their karyotype was analyzed by the spectral karyotyping method. The level of DNA aberrations increased after 3-h exposure to the nanotubes. No appreciable increase in the incidence of aberrant metaphases, micronuclei, and chromosome 1, 6, 8, 11, X, and Y aneuploidy after 24- and 48-h incubation with the nanotubes were detected. Key Words: single-walled carbon nanotubes; human embryonic fibroblasts; spectral karyotyping; DNA aberrations; fluorescent in situ hybridization The use of nanoparticles (NP) is a promising technology for the manufacturing cosmetic articles, drugs, biomimetics, lubricants, and fuel additives, protective and fortifying films, composite, textile, packing materials, lacquers and paints [3]. The creation of nanoindustry is a priority trend of economy in Russia; carbon NP is one of the priorities in the development of materials [1,6]. The possibility of using carbon NP in biotechnology and medicine as biosensors, means for addressed drug delivery, in creation of 3D tissue engineering constructions is demonstrated [10,14]. Sublayers of carbon NP are effective supporting matrix; moreover, they stimulate proliferative activity of cells [7] that can be useful in cell therapy and tissue engineering. Hence, a wide spectrum of technologies and applications of nanomaterials leads to their higher production and their inevitable release into the environment and to contacts with humans. Physicochemical characteristics of NP may differ significantly 1 Medical Genetic Research Center, Russian Academy of Medical Sciences; 2D. I. Ivanovsky Research Institute of Virology, N. F. Gamaleya Federal Research Institute of Epidemiology and Microbiology, Moscow; 3F. F. Erisman Federal Research Center of Hygiene, Mytishchi, Moscow Region; 4N. I. Pirogov Russian National Research Medical University, Ministry of Health of the Russian Federation, Moscow, Russia. Address for correspondence: [email protected]. V. A. Nikitina
from the materials of an equivalent composition, but of larger size, and hence, the data on their cyto- and genotoxicity cannot be directly extrapolated to NP. We studied the genotoxicity of single-walled carbon nanotubes (SWCNT) on human embryonic fibroblasts (HEF) by the methods fit to characterize the possible clastogenic and aneugenic effects of the nanotubes.
MATERIALS AND METHODS DNA breaks emerging within the first 3 h after addition of SWCNT, were evaluated by the DNA comet method. Cytogenetic study of the level of chromosome aberrations was carried out after 24 h, when most cells started division (mitosis metaphase). The aneugenic characteristics of SWCNT were evaluated by analyzing the incidence of micronuclei and aneuploidies by chromosomes 1, 6, 8, 11, X, and Y after the first cell division of HEF after 48 h. Characteristics of SWCNT. SWCNT paste (1 nm in diameter for a solitary nanotube and 10-30 nm in diameter for a bundle of nanotubes) was a kind gift from A. V. Krestinin (Institute of Chemical Physics Problems, Russian Academy of Sciences, Chernogolovka). The paste was autoclaved (1 atm, 120oC,
0007-4888/15/15860812 © 2015 Springer Science+Business Media New York
V. A. Nikitina, A. I. Chausheva, et al.
813
30 min), after which 1% SWCNT in 0.9% NaCl was prepared. Subtoxic concentration of SWCNT corresponding to 1/1000 dilution was selected on the base of previous experiments [2] with the use of MTT test. Characteristics and culturing of HEF cells. The HEF cells were inoculated in a concentration of 100,000 cells/ml and cultured in Eagle’s medium with 10% fetal calf serum (HyClone). The SWCNT preparations were incubated during 3, 24, or 48 h. DNA comet assay. After incubation with SWCNT, the cells were scraped from the bottom of the flask with a silicon spatula, transferred into tubes, and centrifuged. The resultant precipitate was suspended in buffer to the final concentration of 1.5×105 cells/ml. Study by the DNA comet assay (alkaline version) was carried out as described previously [5]. Micropreparations were stained with SYBR Green I fluorescent dye. The DNA comets were analyzed using CASP 1.2.2 software. The percent content of DNA in the comet tail (tail DNA%) served as the indicator of DNA aberrations. Cells with different levels of DNA aberrations were distributed into 5 groups: 0.5, 5.1-10, 10.1-15, 15.1-20 and more tail DNA%. Method for registration of chromosome aberrations. After colchicine treatment, the cells were washed from culture medium and removed from the bottom of the flask with Trypsin–EDTA. The resultant cell suspension was incubated with hypotonic solution (0.05% KCl, 10 min, 37oC) and fixed 3 times (methanol:glacial acetic acid, 3:1), after which the cytogenetic preparations were made by the standard dry air method. The preparations were stained with azure and eosin. Cells with solitary, paired fragments, chromosome and chromatid exchanges were counted. Spectral analysis of chromosomes. Spectral karyotyping (SKY), denaturing, and hybridization of probes and metaphase chromosome preparations
was carried out simultaneously in a hybridizer (ThermoBrite; StatSpin Inc.). A set of specific probes for spectral analysis of chromosomes (Applied Spectral Imaging Inc.) was used. Washing and labeling with antibodies were carried out according to the standard protocol, the images were analyzed by HiSKY 5.5 software (Applied Spectral Imaging Inc.). Micronuclear test. The cells were removed from the flask with Trypsin–EDTA solution, incubated in hypotonic solution (0.55% KCl, 5 min, 4oC), and fixed 3 times (methanol:glacial acetic acid, 3:1). Cell suspensions were applied onto dry cold slides, put on a coolant for 5 min, and dried in the air. The preparations were stained with Acridine Orange, then with DAPI. Interphase FISH analysis. Centromere-specific DNA probes for chromosomes X (DXZ1), Y (DYZ3), 1 (D1Z5), 6 (D6Z1), 8 (D8Z1), and 11 (D11Z1) (Vysis; Abbott) were used. Denaturing, hybridization, and washing were carried out by the standard method. Hybridization mixture for two-color FISH contained samples for three chromosome pairs: X and Y, 1 and 8, 6 and 11. Analysis was carried out as described previously [3], a total of 500 interphase nuclei were analyzed for each chromosome pair three times. Cells with one or three signals by one chromosome and two signals by the other (for pairs 1 and 8, 6 and 11), without signals or with two signals by one chromosome and one signal by the other chromosome (for X and Y pair) were referred to aneuploid. The results were analyzed by Statistica software. Comparative study was carried out by Student’s t and Pearson’s χ2 tests.
RESULTS The HEF cell karyotype was studied by the SKY method before experiment. Twenty-five metaphase
Fig. 1. Spectral analysis of HEF cell karyotype. a) Karyotype (46,XY); b) derivative chromosome t(4:17)(17qter→17p11::4q21→4qter).
Bulletin of Experimental Biology and Medicine, Vol. 158, No. 6, April, 2015 NANOTECHNOLOGIES
814
TABLE 1. Percentage of Cells with Different Levels of DNA Aberrations after Incubation with SWCNT Control (n=239)
SWCNT (n=292)
Parameter abs. Tail DNA %
%
abs.
%
0-5
165
69
192
66
5-10
40
17
47
16
10-15
20
8
22
7.5
15-20
7
3
4
1.5
>20
7
3
27
9
Note. Percent of total number of analyzed cells is shown. p=0.039, df=4, χ2=10.094.
plates were analyzed. The karyotype of 23 plates corresponded to normal male karyotype – 46,XY (Fig. 1, a), while 2 cells contained a derivative chromosome formed by translocation of chromosomes 4 and 17 (Fig. 1, b). The mean level of DNA aberrations in control was 4.58±0.40% (median 2.13); it increased (p=0.04) after 3-h exposure with SWCNT (6.10±0.58 tail DNA%, median 1.97). Analysis of distribution of cells with different levels of DNA aberrations (Table 1) showed that the mean level of aberrations increased after exposure with SWCNT at the expense of higher level of severely damaged cells (more than 20% of tail DNA) vs. control. Comparison of distributions in the control and experiment (df=4, χ2=10.094, p=0.039) confirmed this result. Structural and quantitative mutations were evaluated by chromosomal analysis methods. The incidence of chromosome breaks and translocations was evalua-
ted by analysis of the metaphase plate chromosomes. The level of spontaneous chromosome aberrations was 4.50±0.04% – virtually the same as after incubation with SWCNT – 5.40±0.44% (Table 2). Comparison of the distributions of damaged and intact metaphases in the control and after exposure with SWCNT showed no appreciable differences (df=1, χ2=1.549, p=0.213). The spectrum of chromosome aberrations included mainly chromatid, rarely chromosome fragments and exchanges (Table 2). The incidence of micronuclei, characterizing aneugenic (chromosome lag in the mitosis anaphase) and clastogenic (chromosome break) qualities, did not change after incubation with SWCNT (df=1, χ2=2.21, p=0.14; Fig. 2). The incidence of chromosomes 1, 6, 8, 11, X, and Y aneuploidy was virtually the same in control cultures and in cultures incubated with SWCNT according to analysis with the use of fluorescent centromere samples in interphase nuclei of HEF cells (Table 3). A higher incidence of chromosome Y aneuploidy in comparison with other chromosomes was detected. High incidence of spontaneous DNA and chromosome aberrations in HEF cells is worthy of note; the level of chromosome aberrations in the cells was 1.5-2.0 times higher than in healthy volunteers’ whole blood lymphocytes (2-3% of aberrant metaphases, according to database [4]). No special studies of tissue culture genetic stability have been presented. It seems that a higher changeability in comparison with short-lived lymphocyte culture, traditionally used for evaluation of spontaneous mutagenesis in humans, is characteristic of cultured cells. Hence, the level of DNA aberrations increased in HEF cells incubated with SWCNT, though the incidence of chromosome aberrations, micronuclei, and aneuploidy changed just negligibly. Our results
TABLE 2. Levels of Chromosome Aberrations in Cells Incubated with SWCNT Parameter
Control
SWCNT
950
950
43 (4.5%)
55 (5.44%)
solitary fragments
2.66
3.44
paired fragments
1.78
1.88
chromosome exchanges
0.13
0.12
0
0.18
Total number of metaphases Number of aberrant metaphases Number of structural aberrations/100 cells
chromatid exchanges
Note. *Percent of total level is shown in parentheses. χ2=1.549.
Fig. 2. Incidence of micronuclei in HEF cells. No significant differences detected. df=1, χ2=2.21, p=0.14.
V. A. Nikitina, A. I. Chausheva, et al.
815
TABLE 3. Incidence of Aneuploid Nuclei after SWCNT Exposure Control
SWCNT χ2
Chromosome diploid
aneuploid
diploid
aneuploid
X
980
20
978
22
0.1 (р=0.75)
Y*
955
45
937
63
3.17 (р=0.07)
1
975
25
981
19
0.84 (р=0.36)
8
987
13
985
15
0.14 (р=0.7)
6
983
17
985
15
0.13 (р=0.72)
11
982
18
980
20
0.11 (р=0.74)
Note. *Significant differences in distribution of incidence of chromosome Y aneuploidy in comparison with X, 1, 6, 8, and 11 in control cells and cells exposed to SWCNT.
just partially confirm the data on SWCNT genotoxicity, indicating a higher level of DNA aberrations, micronuclei, and aneuploidy [8,9,11,12]. Induction of ROS, accumulation of LPO products and DNA aberrations (single, double-strand breaks, modified bases, DNA sutures between strands) in the cells and apoptosis triggering are traditionally assumed to be the main mechanisms of the cyto- and genotoxic activities of NP [13]. The possible effects of nanotubes on chromosome deviations in mitosis and on the DNA molecule conformation status are demonstrated [12]. The potential hazards of mutagenic and carcinogenic effects of nanomaterials intended for prospective medical use necessitate further research in genonanotoxicology.
REFERENCES 1. M. V. Alfimov, Ross. Nanotekhnol., 4, Nos. 1-2, 1-8 (2009). 2. I. I. Bobrinetskii, A. S. Seleznyov, I. A. Gaiduchenko, et al., Biofizika, 58, No. 3, 524-530 (2013).
3. N. P. Bochkov, V. A. Nikitina, E. S. Voronina, and N. P. Kuleshov, Bull. Exp. Biol. Med., 148, No. 4, 677-683 (2010). 4. N. P. Bochkov, A. N. Chebotaryov, L. D. Katosova, and V. I. Platonova, Genetika, 37, No. 4, 549-557 (2001). 5. A. K. Zhanataev, V. A. Nikitina, E. S. Voronina, and A. D. Durnev, Prikladn. Toksikol., 2, No. 4, 27-37 (2011). 6. A. V. Krestinin, Ross. Nanotekhnol., 2, Nos. 5-6, 18-23 (2007). 7. R. Ya. Podchernyaeva, I. A. Suetina, G. R. Mikhailova, et al., Vopr. Virusol., 57, No. 5, 46-48 (2012). 8. J. Cveticanin, G. Joksic, A. Leskovac, et al., Nanotechnology, 21, No. 1, doi: 10.1088/0957-4484/21/1/015102 (2010). 9. N. R. Jacobsen, G. Pojana, P. White, et al., Environ. Mol. Mutagen., 49, No. 6, 476-487 (2008). 10. Y. Lin, S. Taylor, H. Li, et al., J. Mater. Chem., No. 14, 527541 (2004). 11. L. Migliore, D. Saracino, A. Bonelli, et al., Environ. Mol. Mutagen., 51, No. 4, 294-303 (2010). 12. L. M. Sargent, A. A. Shvedova, A. F. Hubbs, et al., Environ. Mol. Mutagen., 50, No. 8, 708-717 (2009). 13. N. Singh, B. Manshian, G. J. Jenkins, et al., Biomaterials, 30, Nos. 23-24, 3891-3941 (2009). 14. F. Valentini, M. Carbone, and G. Paleschi, Anal. Bioanal. Chem., 405, Nos. 2-3, 451-465 (2013).
