Testing Strategies of the In Vitro Micronucleus Assay for the Genotoxicity Assessment of Nanomaterials in BEAS-2B Cells
Abstract
:1. Introduction
2. Materials and Methods
2.1. Preparation of NMs
2.2. Characterisation of NMs
2.3. Cell Cultivation and Treatment Conditions
2.4. Micronucleus Analysis
2.5. Transmission Electron Microscopy
2.6. Statistical Analysis
3. Results
3.1. Characterisation of NMs
3.2. Transmission Electron Microscopy
3.3. Micronucleus Analysis
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
Abbreviations
References
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Nanomaterial | Details | References | ||||
---|---|---|---|---|---|---|
Type of Material | NM Code | Shape | Size | Aggregation (Size Range) | Other Information | |
TiO2 | NM-101 | Spherical or ellipsoidal | 5 nm | 10–170 nm 95.2% up to 100 nm 77.3% up to 50 nm | Anatase form | [23] |
TiO2 | NM-103 | Ellipsoidal | 20–100 nm | 40–400 nm 51.8% up to 100 nm 12.7% up to 50 nm | Rutile form | [23] |
SiO2 | NM-200 | Spherical or ellipsoidal | 10–20 nm | 15–650 nm 88.7% up to 100 nm 69.8% up to 50 nm | Precipitated | [24] |
Ag | NM-300K | Round, triangular or trapezium | <20 nm | Low risk a | [25] | |
Ag | NM-302 | Rods | 100–200 nm (width) 5–10 µm (length) | Aggregates of 5–10 particles (most common) up to thousands of particles (size > 10 µm) (rare) | [26] |
Nanomaterials | Properties (Mean ± SD) | ||||
---|---|---|---|---|---|
Type of Material | NM Code | Concentration | Hydrodynamic Size [nm] | Polydispersity Index | Zeta-Potential [mV] |
TiO2 | NM-101 | 2.56 mg/mL | 481.82 ± 16.40 | 0.35 ± 0.04 | n. m. |
NM-103 | 2.56 mg/mL | 286.75 ± 21.69 | 0.36 ± 0.01 | n. m. | |
SiO2 | NM-200 | 2.56 mg/mL | 214.07 ± 5.83 | 0.30 ± 0.03 | n. m. |
Ag | NM-300K | 2.56 mg/mL | 75.66 ± 0.81 | 0.28 ± 0.01 | n. m. |
NM-302 | 2.56 mg/ml | 451.07 ± 59.21 | 0.44 ± 0.11 | n. m. |
Nanomaterials | Properties | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Type of Material | NM Code | Conc. | Hydrodynamic Size [nm] (Mean ± SD) | Polydispersity Index (Mean ± SD) | Zeta-Potential [mV] (Mean ± SD) | ||||||
0 h | 28 h | 48 h | 0 h | 28 h | 48h | 0 h | 28 h | 48 h | |||
TiO2 | NM-101 | 1 µg/mL | 328.08 ± 97.08 | 535.05 ± 185.24 | 1226.57 ± 83.31 | 0.46 ± 0.06 | 0.56 ± 0.12 | 0.36 ± 0.08 | −14.35 ± 0.66 | −14.89 ± 1.36 | −17.64 ± 0.81 |
10 µg/mL | 491.30 ± 54.83 | 417.43 ± 92.98 | 364.04 ± 72.42 | 0.37 ± 0.06 | 0.44 ± 0.06 | 0.46 ± 0.08 | −13.43 ± 0.92 | −12.35 ± 1.33 | −13.10 ± 0.87 | ||
25 µg/mL | 496.59 ± 17.11 | 422.23 ± 38.99 | 449.18 ± 72.40 | 0.35 ± 0.02 | 0.49 ± 0.05 | 0.50 ± 0.06 | −14.04 ± 1.48 | −12.98 ± 1.14 | −15.00 ± 1.63 | ||
TiO2 | NM-103 | 1 µg/mL | 173.38 ± 22.29 | 223.65 ± 69.07 | 201.06 ± 17.45 | 0.28 ± 0.08 | 0.26 ± 0.04 | 0.31 ± 0.06 | −11.71 ± 0.97 | −10.90 ± 1.16 | −11.17 ± 0.79 |
10 µg/mL | 179.03 ± 7.69 | 184.20 ± 8.92 | 196.69 ± 12.91 | 0.25 ± 0.04 | 0.20 ± 0.06 | 0.25 ± 0.09 | −11.65 ± 0.87 | −12.39 ± 1.38 | −12.70 ± 0.73 | ||
25 µg/mL | 176.80 ± 7.74 | 198.70 ± 10.20 | 203.22 ± 40.72 | 0.26 ± 0.04 | 0.26 ± 0.04 | 0.27 ± 0.05 | −13.21 ± 1.11 | −12.12 ± 0.73 | −13.51 ± 1.38 | ||
SiO2 | NM-200 | 1 µg/mL | 169.12 ± 19.18 | 305.74 ± 131.54 | 2317.11 ± 581.67 | 0.47 ± 0.08 | 0.57 ± 0.09 | 0.97 ± 0.06 a | −11.59 ± 1.27 | −18.76 ± 2.31 | −14.85 ± 1.56 |
2.5 µg/mL | 196.97 ± 18.18 | 269.07 ± 97.76 | 473.70 ± 238.19 | 0.44 ± 0.03 | 0.61 ± 0.11 a | 0.59 ± 0.08 | −12.32 ± 1.32 | −13.54 ± 1.65 | −10.99 ± 2.75 | ||
5 µg/mL | 214.07 ± 5.83 | 321.40 ± 59.21 | 1518.18 ± 98.06 | 0.03 ± 0.03 | 0.55 ± 0.12 | 0.65 ± 0.09 a | −12.98 ± 1.31 | −12.95 ± 1.76 | −12.87 ± 1.23 | ||
Ag | NM-300K | 1 µg/mL | 87.35 ± 28.13 | 95.78 ± 8.49 | 128.97 ± 31.27 | 0.37 ± 0.07 | 0.33 ± 0.07 | 0.46 ± 0.06 | −12.43 ± 1.46 | −12.29 ± 1.54 | −13.71 ± 1.99 |
2.5 µg/mL | 83.38 ± 1.44 | 121.82 ± 38.25 | 137.31 ± 31.75 | 0.31 ± 0.03 | 0.31 ± 0.07 | 0.45 ± 0.08 | −12.70 ± 1.34 | −13.08 ± 1.59 | −13.80 ± 2.16 | ||
5 µg/mL | 92.86 ± 2.51 | 208.28 ± 10.34 | 767.25 ± 81.01 | 0.28 ± 0.03 | 0.42 ± 0.02 | 0.66 ± 0.12 a | −11.56 ± 1.07 | −12.78 ± 1.53 | −21.11 ± 1.64 | ||
Ag | NM-302 | 1 µg/mL | 1305.16 ± 1118.92 | 191.39 ± 66.06 | 317.95 ± 259.75 | 0.78 ± 0.25 a | 0.53 ± 0.11 | 0.60 ± 0.18 a | −14.20 ± 1.57 | −13.07 ± 1.77 | −13.31 ± 1.70 |
10 µg/mL | 1224.80 ± 261.61 | 1469.97 ± 679.78 | 1130.63 ± 702.44 | 0.85 ± 0.14 a | 0.95 ± 0.09 a | 0.79 ± 0.24 a | −12.81 ± 0.86 | −13.75 ± 1.15 | −12.83 ± 1.13 | ||
25 µg/mL | 816.42 ± 148.07 | 1094.03 ± 611.9 | 1456.28 ± 599.52 | 0.70 ± 0.09 a | 0.83 ± 0.16 a | 0.88 ± 0.12 a | −15.05 ± 1.50 | −13.97 ± 1.04 | −14.18 ± 1.80 |
Nanomaterials | Properties | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Type of Material | NM Code | Conc. | Hydrodynamic Size [nm] (mean ± SD) | Polydispersity Index (mean ± SD) | Zeta-Potential [mV] (mean ± SD) | ||||||
0 h | 28 h | 48h | 0 h | 28 h | 48h | 0 h | 28 h | 48 h | |||
TiO2 | NM-101 | 1 µg/mL | 338.52 ± 57.40 | 292.89 ± 87.35 | 301.13 ± 52.18 | 0.48 ± 0.06 | 0.43 ± 0.06 | 0.49 ± 0.08 | −13.80 ± 1.32 | −13.34 ± 1.11 | −14.15 ± 1.78 |
10 µg/mL | 457.29 ± 29.64 | 324.67 ± 48.30 | 272.77 ± 15.09 | 0.35 ± 0.03 | 0.332 ± 0.07 | 0.29 ± 0.06 | −12.03 ± 0.84 | −12.02 ± 1.06 | −13.86 ± 1.21 | ||
25 µg/mL | 472.23 ± 15.35 | 359.9 ± 2298 | 327.09 ± 42.38 | 0.29 ± 0.03 | 0.32 ± 0.05 | 0.36 ± 0.07 | −13.18 ± 1.42 | −13.49 ± 1.26 | −13.55 ± 0.53 | ||
TiO2 | NM-103 | 1 µg/mL | 157.78 ± 9.13 | 171.43 ± 5.80 | 176.11 ± 6.40 | 0.25 ± 0.05 | 0.22 ± 0.02 | 0.21 ± 0.03 | −12.13 ± 1.31 | −11.74 ± 1.14 | −12.62 ± 0.98 |
10 µg/mL | 170.34 ± 11.03 | 190.71 ± 27.24 | 198.16 ± 10.98 | 0.24 ± 0.02 | 0.22 ± 0.07 | 0.25 ± 0.05 | −13.55 ± 0.91 | −12.69 ± 1.06 | −14.44 ± 1.09 | ||
25 µg/mL | 176.98 ± 12.53 | 177.29 ± 4.37 | 187.26 ± 7.08 | 0.27 ± 0.04 | 0.20 ± 0.04 | 0.22 ± 0.05 | −12.83 ± 1.34 | −13.29 ± 0.71 | −13.41 ± 1.68 | ||
SiO2 | NM-200 | 1 µg/mL | 179.87 ± 9.75 | 171.54 ± 20.58 | 345.68 ± 33.06 | 0.48 ± 0.03 | 0.48 ± 0.13 | 0.61 ± 0.15 a | −12.13 ± 1.66 | −11.67 ± 0.80 | −12.69 ± 1.84 |
2.5 µg/mL | 228.86 ± 15.24 | 443.91 ± 128.20 | 261.11 ± 60.00 | 0.45 ± 0.04 | 0.61 ± 0.13 a | 0.52 ± 0.07 | −12.19 ± 1.62 | −12.59 ± 1.07 | −11.44 ± 1.01 | ||
5 µg/mL | 239.98 ± 7.19 | 271.98 ± 13.32 | 306.87 ± 103.28 | 0.39 ± 0.03 | 0.53 ± 0.08 | 0.49 ± 0.08 | −11.33 ± 0.86 | −12.42 ± 1.36 | −11.64 ± 1.07 | ||
Ag | NM-300K | 1 µg/mL | 98.03 ± 16.15 | 115.68 ± 29.75 | 112.68 ± 11.32 | 0.31 ± 0.08 | 0.33 ± 0.05 | 0.33 ± 0.06 | −12.34 ± 1.04 | −9.54 ± 1.08 | −10.39 ± 1.27 |
2.5 µg/mL | 130.07 ± 3.38 | 115.33 ± 12.71 | 122.95 ± 4.81 | 0.45 ± 0.04 | 0.29 ± 0.05 | 0.30 ± 0.04 | −11.67 ± 1.48 | −11.36 ± 1.23 | −10.66 ± 1.53 | ||
5 µg/mL | 88.23 ± 3.68 | 188.85 ± 3.75 | 223.93 ± 8.09 | 0.30 ± 0.05 | 0.26 ± 0.01 | 0.38 ± 0.01 | −13.05 ± 1.07 | −11.65 ± 0.93 | −9.94 ± 1.25 | ||
Ag | NM-302 | 1 µg/mL | 1139.19 ± 500.85 | 142.20 ± 9.56 | 3038.32 ± 551.73 | 0.84 ± 0.13 a | 0.47 ± 0.08 | 0.81 ± 0.09 a | −14.18 ± 0.99 | −12.70 ± 0.90 | −11.92 ± 0.88 |
10 µg/mL | 649.89 ± 63.78 | 485.20 ± 241.45 | 266.27 ± 81.81 | 0.63 ± 0.07 | 0.68 ± 0.11 a | 0.54 ± 0.13 | −14.07 ± 0.84 | −14.83 ± 1.37 | −14.08 ± 0.80 | ||
25 µg/mL | 527.47 ± 26.76 | 704.09 ± 280.04 | 580.01 ± 398.89 | 0.47 ± 0.03 | 0.68 ± 0.13 a | 0.59 ± 0.2 1a | −14.03 ± 1.08 | −15.01 ± 1.54 | −14.71 ± 1.57 |
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Cervena, T.; Rossnerova, A.; Zavodna, T.; Sikorova, J.; Vrbova, K.; Milcova, A.; Topinka, J.; Rossner, P., Jr. Testing Strategies of the In Vitro Micronucleus Assay for the Genotoxicity Assessment of Nanomaterials in BEAS-2B Cells. Nanomaterials 2021, 11, 1929. https://doi.org/10.3390/nano11081929
Cervena T, Rossnerova A, Zavodna T, Sikorova J, Vrbova K, Milcova A, Topinka J, Rossner P Jr. Testing Strategies of the In Vitro Micronucleus Assay for the Genotoxicity Assessment of Nanomaterials in BEAS-2B Cells. Nanomaterials. 2021; 11(8):1929. https://doi.org/10.3390/nano11081929
Chicago/Turabian StyleCervena, Tereza, Andrea Rossnerova, Tana Zavodna, Jitka Sikorova, Kristyna Vrbova, Alena Milcova, Jan Topinka, and Pavel Rossner, Jr. 2021. "Testing Strategies of the In Vitro Micronucleus Assay for the Genotoxicity Assessment of Nanomaterials in BEAS-2B Cells" Nanomaterials 11, no. 8: 1929. https://doi.org/10.3390/nano11081929
APA StyleCervena, T., Rossnerova, A., Zavodna, T., Sikorova, J., Vrbova, K., Milcova, A., Topinka, J., & Rossner, P., Jr. (2021). Testing Strategies of the In Vitro Micronucleus Assay for the Genotoxicity Assessment of Nanomaterials in BEAS-2B Cells. Nanomaterials, 11(8), 1929. https://doi.org/10.3390/nano11081929