No evidence of the long-term in vitro toxicity of Aeroxide P25 TiO2 nanoparticles in three mammalian cell lines despite the initial reduction of cellular mitochondrial activity

Medium	Hydrodynamic diameter (nm)	Polydispersity index (PDI)	Zeta potential 7.4 (mV)
Water	356 ± 1	0.457 ± 0.04	–15.3 ± 0.5
EMEM	246 ± 1	0.193 ± 0.07	–25.5 ± 0.6
F12	295 ± 3	0.220 ± 0.05	–28.7 ± 2.5
DMEM	300 ± 2	0.280 ± 0.07	–27.5 ± 0.2

1 The data provided are in the form mean ± SD.

n denotes the number of experiments; n = 3.

SSB and base damage recognized by FPG in A549, HepG2 or HT29 cells treated with Aeroxide P25 TiO₂ NP for 2 h (left panel) and 24 h (right panel) at concentrations indicated. Numbers (1, 2) denote results of Tukey’s test *post hoc* analysis, indicating a significant difference of the means from the control when NP concentration was considered (1) or a significant difference of the means between the same NP concentration, but different treatment times (2).

Table 2.

Frequency of micronuclei and mean number of γ-H2AX foci per nucleus after 2 h and 24 h treatment with TiO₂ NP concentrations indicated, and with the effect of X-irradiation with 2 Gy shown for comparison

Micronuclei frequency
Treatment	Dose	Cell line/time of treatment
		A549		HepG2		HT29
		2 h	24 h	2 h	24 h	2 h	24 h
Control		22.9 ± 9.5	31.5 ± 2.0	34.6 ± 3.4	26.3 ± 7.8	5.9 ± 3.2	7.5 ± 1.8
X-ray	2 Gy	80.1 ± 5.2^a		155.2 ± 7.7^a		45.5 ±0.3^a
TiO₂ NP 21 nm	10 μg/mL	33.2 ± 6.9	26.8 ± 7.5	22.3 ± 8.8	27.0 ± 9.5	6.8 ± 1.0	8.5 ± 6.6
	50 μg/mL	22.5 ± 9.6	25.2 ± 3.7	24.6 ± 4.5	23.0 ± 4.4	5.5 ± 0.9	7.8 ± 1.6
	100 μg/mL	25.5 ± 5.8	30.0 ± 8.2	24.5 ± 12.5	23.5 ± 6.4	6.1 ± 2.8	10.0 ± 7.2
The mean number of γ-H2AX foci per nucleus
Treatment	Dose	Cell line/time of treatment
		A549		HepG2		HT29
		2 h	24 h	2 h	24 h	2 h	24 h
Control		10.8 ± 5.7	10.7 ± 3.0	23.6 ± 10.8	7.2 ± 2.1	25.5 ± 10.1	26.4 ± 10.1
X-ray	2 Gy	78.2 ± 10.2^a		87.2 ± 10.0”		87.9 ± 3.4^a
TiO₂ NP 21 nm	10 μg/mL	11.2 ± 6.8	10.5 ± 3.1	21.1 ± 6.0	9.7 ± 4.0	25.4 ± 10.0	25.0 ± 9.4
	50 μg/mL	10.3 ± 3.5	7.8 ± 1.5	19.3 ± 5.6	11.4 ± 2.7	26.6 ± 6.8	23.7 ± 8.8
	100 μg/mL	10.6 ± 4.1	6.4 ± 2.5	18.3 ± 8.0	9.5 ± 3.2	28.0 ± 5.0	24.5 ± 10.5

1 The data provided are in the form mean ± SD.

n denotes the number of experiments; n = 3.

1a denotes statistically important difference from control (Student’s t-test, P < 0.05).

Table 3.

The extent of apoptosis after 2 h and 24 h treatment with TiO₂ NP at indicated concentrations

Treatment	Dose	Cell line/time of treatment
		A549		HepG2		HT29
		2h	24 h	2h	24 h	2h	24 h
Control	Viable	99.16 ± 0.14	98.36 ± 0.16	97.63 ± 0.29	96.34 ± 0.43	98.03 ± 0.55	97.04 ± 0.69
	Early apoptosis	0.54 ± 0.21	1.34 ± 0.26	2.20 ± 0.30	3.22 ± 0.29	1.80 ± 0.56	2.61 ± 0-43
	Late apoptosis and necrosis	0.30 ± 0.10	0.30 ± 0.08	0.17 ± 0.15	0.44 ± 0.17	0.17 ± 0.06	0.35 ± 0.09
Staurosporine/24 h^b	Viable	32.48 ± 8.88^a		23.70 ± 5.33^a		20.20 ± 3.20^a
	Early apoptosis	7.98 ± 2.33^a		48.20 ± 3.22^a		58.03 ± 4.98^a
	Late apoptosis and necrosis	59.54 ± 3.99^a		28.10 ± 2.56^a		21.77 ± 2.37^a
TiO₂ NP 10 mg/mL	Viable	93.07 ± 0.81^a	93.30 ± 1.20^a	93.20 ± 0.26^a	92.93 ± 0.64^a	93.13 ± 0.25^a	92.50 ± 1.46^a
	Early apoptosis	5.10 ± 0.44^a	4.70 ± 0.75^a	5.03 ± 1.03	4.53 ± 0.40^a	4.43 ± 0.75	4.17 ± 0.79
	Late apoptosis and necrosis	1.83 ± 0.40^a	2.00 ± 0.46^a	1.77 ± 0.81	2.53 ± 0.26^a	2.43 ± 0.50^a	3.33 ± 0.81^a
TiO₂ NP 50 mg/mL	Viable	92.10 ± 0.80^a	93.93 ± 1.75^a	93.97 ± 0.65^a	91.13 ± 1.37^a	93.17 ± 0.78^a	91.83 ± 1.60^a
	Early apoptosis	5.97 ± 0.76^a	3.83 ± 1.33^a	3.97 ± 0.61	6.00 ± 1.15^a	3.70 ± 0.60	5.17 ± 1.40^a
	Late apoptosis and necrosis	1.93 ± 0.15^a	2.23 ±0.42^a	2.07 ±0.15^a	2.87 ± 0.35^a	3.13 ±0.25^a	3.00 ± 0.26^a
TiO₂NP 100 mg/mL	Viable	93.63 ± 1.20^a	91.87 ± 1.10^a	93.73 ± 0.64^a	90.87 ± 0.96^a	92.23 ± 1.46^a	90.57 ± 0.23^a
	Early apoptosis	3.97 ± 0.75^a	5.90 ± 1.47^a	4.27 ± 0.40^a	6.33 ± 0.91^a	4.70 ± 0.79^a	6.17 ± 0.65^a
	Late apoptosis and necrosis	2.40 ± 0.46^a	2.23 ± 0.40^a	2.00 ± 0.26^a	2.80 ± 0.26^a	3.07 ± 0.81^a	3.27 ± 0.47^a

1 The data provided are in the form mean ± SD.

n denotes the number of experiments; n = 3.

1a denotes statistically important difference from control (Student’s t-test, P < 0.05).

1b denotes the effect of staurosporine being shown for comparison (positive control).

Table 4.

Caspase 3 activity after 2 h and 24 h treatment with TiO₂ NP at indicated concentrations

Treatment	Cell line/time of treatment
	A549		HepG2		HT29
	2 h	24 h	2 h	24 h	2 h	24 h
Control	0.12 ±0.05	0.05 ±0.01	1.02 ±0.03	1.42 ±0.02	0.26 ±0.02	0.42 ±0.03
TiO₂ NP 10 mg/mL	0.10 ± 0.02	0.06 ± 0.01	0.98 ± 0.02	1.43 ± 0.09	0.31 ± 0.01	0.41 ± 0.06
TiO₂ NP 50 mg/mL	0.13 ± 0.02	0.07 ± 0.01	1.08 ± 0.07	1.37 ± 0.05	0.51 ± 0.05^a	0.63 ±0.05^a
TiO₂ NP 100 mg/mL	0.11 ± 0.01	0.07 ± 0.01	1.39 ± 0.06^a	1.39 ± 0.03	0.37 ± 0.02^a	0.55 ± 0.04^a
Positive control: Jurkat cells treated with 1 mM staurosporine for 24 h : 39.89 ± 3.82^a

1 The effect of staurosporine is shown for comparison.

The data provided are in the form mean ± SD.

n denotes the number of experiments; n = 3.

1a denotes statistically important difference from control (Student’s t-test, P < 0.05).

Mitochondrial activity (MTT assay) of cells treated with Aeroxide P25 TiO₂ NP for 24 h and measured 72 h after treatment expressed as a percent of the control (untreated cells). The asterisk denotes a statistically significant difference from the control. Two-way ANOVA with Tukey’s HDS *post hoc* comparison. P < 0.05.

Short-term survival of cells treated with Aeroxide P25 TiO₂ NP for 24 h, as measured by neutral red assay. The asterisk denotes a statistically significant difference from the control. Two-way ANOVA with Tukey’s HDS *post hoc* comparison. P < 0.05.

Cloning ability determination of A549, HT29 and HepG2 cells growing in the presence of Aeroxide P25 TiO₂ NP.

Generation of ROS in HepG2, A549 and HT29 cells incubated with Aeroxide P25 TiO₂ NP. H₂O₂ in a concentration of 1 mM served as a positive control. The data provided are in the form mean ± SD. n denotes the number of experiments; n = 3. The asterisk denotes statistically significant difference from unexposed control, P < 0.05.

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DOI: https://doi.org/10.2478/nuka-2024-0002 | Journal eISSN: 1508-5791 (formerly 0029-5922) | Journal ISSN: 0029-5922

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Language: English

Page range: 13 - 22

Submitted on: Mar 8, 2023

Accepted on: Nov 27, 2023

Published on: Feb 23, 2024

Published by: Institute of Nuclear Chemistry and Technology

In partnership with: Paradigm Publishing Services

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© 2024 Sylwia Męczyńska-Wielgosz, Teresa Bartłomiejczyk, Iwona Grądzka, Sylwester Sommer, Aneta Węgierek-Ciuk, Anna Lankoff, Katarzyna Sikorska, Maria Wojewódzka, Małgorzata Dobrzyńska, Marcin Kruszewski, published by Institute of Nuclear Chemistry and Technology
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

Volume 69 (2024): Issue 1 (March 2024)

No evidence of the long-term in vitro toxicity of Aeroxide P25 TiO2 nanoparticles in three mammalian cell lines despite the initial reduction of cellular mitochondrial activity

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Fig. 1.

Table 2.

Table 3.

Table 4.

Fig. 2.

Fig. 3.

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Fig. 5.

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