Aneuploidogenic effects and DNA oxidation induced in vitro by differently sized gold nanoparticles

Figures & data

Table 1 Au NP characterization

Au NP nominal size	STEM	DLS in native solution			CPS in native solution			CPS in MEM
	Mean ± SD (nm)	Mean ± SD (nm)	PDI	Z-pot (mV)	Mean (nm)	Half-width (nm)	PDI	Mean (nm)	Half-width (nm)	PDI
5 nm	3.3±1.7	7.1±2.1	0.097	−26±11	6.6	2.2	1.37	3.4	1.5	5
15 nm	12.0±1.0	17.0±4.7	0.028	−30±12	12.9	1.7	1.04	7.4	1.3	2.3

Notes: Mean size distribution ± SD of 5 nm and 15 nm Au NPs measured in native solution by STEM, DLS, and CPS. PDI and Z-potential values are also included. Values are expressed as mean of three measurements for DLS and CPS, and for STEM analysis of at least 300 NPs. The mean size distribution of 5 nm and 15 nm Au NPs obtained by CPS was measured in MEM after 72 hours of incubation at 37°C. Z-potential measurements of the as-synthesized Au NPs were performed at the physiological pH range of 6.5–7.5.

Abbreviations: Au NP, gold nanoparticle; CPS, centrifugal particle sedimentation; DLS, dynamic light scattering; MEM, minimal essential medium; PDI, polydispersity index; SD, standard deviation; STEM, scanning transmission electron microscopy; Z-pot, Z-potential.

Figure 1 Cell viability, evaluated by MTT assay, in PBL and Raw264.7 after 2 hours and 24 hours of exposure to 5 nm and 15 nm Au NPs.

Notes: PBL following 24 hours treatment with Au NP 15 nm showed a dose-effect relationship (P=0.01). Raw264.7 following 2 hours treatment with 5 nm Au NPs showed a dose-effect relationship (P=0.04) as well as after 24 hours treatment to 5 nm and 15 nm Au NPs (P=0.03 and P=0.016, respectively). Data show the percent of viable cells normalized to untreated control (±SEM, n=9). Statistically significant differences from the C− were determined by Student’s t-test (*P<0.05; **P<0.01; ***P<0.001).

Abbreviations: Au NP, gold nanoparticle; C+, positive control (10 μM hydrogen peroxide); C−, negative (untreated) control; h, hours; MTT, methylthiazol tetrazolium; PBL, peripheral blood lymphocytes; SEM, standard error of the mean.

Table 2 Cytostasis and cytotoxicity evaluated by CBMN Cyt assay

	CBPI		Mitotic index		Apoptotic index		Necrotic index
	PBL	Raw264.7	PBL	Raw264.7	PBL	Raw264.7	PBL	Raw264.7
C−	1.88±0.02	1.85±0.04	6.5±0.1	5.6±0.2	0.7±0.1	1.7±0.2	0.2±0.2	0.9±0.3
C+	1.17±0.13***	1.41±0.06***	0.3±0.2***	2.3±0.6***	4.7±0.3***	4.6±0.5***	2.5±0.4***	4.0±0.4
Au NP 5 nm
0.1 μg/mL	1.55±0.02***	1.62±0.06**	3.9±0.2***	4.0±0.4***	1.2±0.2*	2.0±0.5	1.6±0.3***	3.4±0.4***
1 μg/mL	1.54±0.05***	1.62±0.05**	4.6±0.6**	3.9±0.4***	1.9±0.5**	1.2±0.3	2.4±0.3***	2.1±0.5**
10 μg/mL	1.47±0.02***	1.57±0.05**	4.4±0.3***	3.6±0.3***	1.6±0.2**	1.8±0.5	2.6±0.2***	2.9±0.2***
100 μg/mL	1.36±0.05***	1.51±0.07***	2.8±0.4***	3.1±0.4***	1.8±0.4**	2.6±0.2***	3.0±0.6***	4.3±0.6***
Au NP 15 nm
0.1 μg/mL	1.70±0.03*	1.68±0.04**	4.9±0.5**	3.7±0.3***	1.5±0.1**	1.7±0.1	0.6±0.2*	1.4±0.2*
1 μg/mL	1.57±0.02***	1.61±0.01***	4.0±0.6***	2.8±0.2***	1.9±0.2**	2.3±0.3*	0.9±0.1**	1.7±0.3**
10 μg/mL	1.56±0.05***	1.60±0.04***	3.5±0.3***	2.5±0.2***	3.5±0.3***	2.8±0.4**	1.6±0.2***	2.5±0.3***
100 μg/mL	1.34±0.04***	Toxic	1.1±0.6***	Toxic	9.1±0.2***	Toxic	3.7±0.6***	Toxic

Notes: CBPI and mitotic (cytostasis), apoptotic, and necrotic (cytotoxicity) indices scored by CBMN Cyt assay. CBPI of PBL exposed to 5 nm and 15 nm Au NPs decreased with a dose-dependent relationship (P=0.027 and P=0.02, respectively) as well as for Raw264.7 exposed to 5 nm Au NPs (P=0.04). Dose-dependent decrease in the mitotic index was observed in PBL exposed to 15 nm Au NPs (P=0.029) and in Raw264.7 exposed to 5 nm and 15 nm Au NPs (P=0.033 and P=0.05, respectively). Both PBL and Raw264.7 exposed to 15 nm Au NPs showed dose-dependent apoptotic index increase (P=0.049 and P=0.05, respectively); the necrotic index induced a dose-dependent effect in PBL exposed to 5 nm and 15 nm Au NPs (P=0.016 and P=0.03, respectively) and in Raw264.7 exposed to 15 nm Au NPs (P=0.017). 100 μg/mL 15 nm Au NPs resulted in illegible slides. Statistically significant differences from the control were determined by the Student’s t-test (*P<0.05; **P<0.01; ***P<0.001). Data represent the mean ± SEM. PBL: n=4; Raw264.7: n=6.

Abbreviations: Au NP, gold nanoparticle; C+, positive control (0.1 μg/mL MMC); C−, negative (untreated) control; CBMN Cyt, cytokinesis-block micronucleus cytome; CBPI, cytokinesis block proliferation index; MMC, mitomycin C; PBL, peripheral blood lymphocytes; SEM, standard error of the mean.

Table 3 Genotoxicity evaluated by CBMN Cyt assay in PBL and Raw264.7 cells exposed to increasing mass concentrations of Au NPs

	MN		NPB		NBUD
	PBL	Raw264.7	PBL	Raw264.7	PBL	Raw264.7
C−	3.25±0.75	2.3±0.5	0.5±0.5	1.2±0.4	0±0	0.5±0.5
C+	43.3±7.2***	32.4±5.6***	14.4±0.8***	13.4±1.0**	2.8±0.5***	7.0±1.5***
Au NP 5 nm
0.1 μg/mL	5.0±1.4	5.1±1.1	2.2±0.5**	1.8±0.5	1.5±0.5**	1.3±0.6
1 μg/mL	7.0±2.0	4.5±1.2	2.2±0.2**	1.4±0.8	4.3±0.3***	1.0±0.8
10 μg/mL	8.5±0.35*	5.4±1.4	2.0±0.4**	1.4±0.8	2.5±0.4***	2.0±0.8
100 μg/mL	12.5±0.35**	7.4±1.1**	3.2±0.6**	1.8±0.4	6.7±0.9***	1.3±0.4
Au NP 15 nm
0.1 μg/mL	9.2±1.2*	17.5±1.5**	2.0±0.5**	4.2±1.2*	0.5±0.5	5.5±0.5***
1 μg/mL	12.5±0.5**	25.0±2.0***	2.8±0.7**	4.8±0.5*	2.5±0.5***	7.0±1.0***
10 μg/mL	14.3±1.2**	27.5±2.5***	2.8±1.0**	4.8±0.8*	5.5±0.7***	7.5±1.5***
100 μg/mL	22.4±2.3***	Toxic	3.2±1.5**	Toxic	12±1***	Toxic

Notes: MN, NPB, and NBUD frequencies scored by CBMN Cyt assay after exposure of PBL and Raw264.7 to increasing mass concentrations of Au NPs (0.1–100 μg/mL). In PBL, 5 nm and 15 nm Au NPs induced dose-dependent effect in MN (P=0.0014 and P=0.03, respectively), NPB (P=0.012 and P=0.033; respectively), and NBUD frequency (P=0.05 and P=0.02, respectively). MN frequency of Raw264.7 exposed to 15 nm Au NPs showed a dose-dependent relationship (P=0.05). Statistically significant differences from the control were determined by the Student’s t-test (*P<0.05; **P<0.01; ***P<0.001). Data represent the mean ± SEM (PBL: n=4; Raw264.7: n=6).

Abbreviations: Au NP, gold nanoparticle; C+, positive control (0.1 μg/mL mitomycin C); C−, negative (untreated) control; CBMN Cyt, cytokinesis-block micronucleus cytome; MN, micronuclei; NBUD, nuclear buds; NPB, nucleoplasmic bridges; PBL, peripheral blood lymphocytes; SEM, standard error of the mean.

Figure 2 FISH analysis in PBL and Raw264.7 after exposure to 5 nm and 15 nm Au NPs.

Notes: Using pancentromeric probes applied to the CBMN Cyt slides, the centromeric spots were analyzed and the exposure of Raw264.7 to 15 nm Au NPs showed dose-dependent (P=0.01) aneuploidogenic events. Data are shown as mean ± SEM (PBL: n=4; Raw264.7: n=6). Student’s t-test: *P<0.05; **P<0.01; ***P<0.001.

Abbreviations: Au NP, gold nanoparticle; C+, positive control (MMC 0.1 μg/mL); C−, negative (untreated) control; CBMN Cyt, cytokinesis-block micronucleus cytome; FISH, fluorescence in situ hybridization; h, hours; MN C+, micronuclei centromere +ve; MN C−, micronuclei centromere -ve; PBL, peripheral blood lymphocytes; SEM, standard error of the mean.

Figure 3 Comet assay to evaluate primary DNA damage/breakage effects in PBL and Raw264.7.

Notes: The 2-hour treatments with 5 nm and 15 nm Au NPs showed a dose-effect relationship in PBL (P=0.05 and P=0.002) and in Raw264.7 (P=0.0014 and P=0.03, respectively). The 24-hour exposure to 5 nm and 15 nm Au NPs showed a dose-effect relationship in Raw264.7 (P=0.0016 and P=0.004, respectively), but only 15 nm Au NPs in PBL (P=0.006). Data are shown as mean ± SEM (PBL: n=4; Raw264.7: n=6) of percent DNA in tail. Student’s t-test: 2-hour data, P<0.01; 24-hour data, P<0.001.

Abbreviations: Au NP, gold nanoparticle; C+, positive control (10 μM hydrogen peroxide); C−, negative (untreated) control; DNA, deoxyribonucleic acid; h, hours; PBL, peripheral blood lymphocytes; SEM, standard error of the mean.

Figure 4 Oxidative DNA damage evaluated using the enzyme-modified comet assay in PBL and Raw264.7.

Notes: Dose-dependent pyrimidines oxidation was detected in PBL after 24 hours of exposure to 5 nm and 15 nm Au NPs (P=0.05 and P=0.028, respectively). In Raw264.7 the pyrimidines oxidation, detected by the EndoIII enzyme, showed a dose-dependent effect after 2 hours of exposure to 15 nm Au NPs (P=0.035) and at 24 hours to 5 nm and 15 nm Au NPs (P=0.009 and P=0.006, respectively). By Fpg, dose-dependent purines oxidation was observed in PBL after 2 hours of exposure to 5 nm and 15 nm Au NPs (P=0.009 and P=0.007, respectively), as well as after 24 hours of exposure to 5 nm Au NPs (P=0.014). In Raw264.7, a dose-effect relationship was detected following 2 hours of exposure to 15 nm Au NPs (P=0.01) and 24 hours of incubation in the presence of 5 nm Au NPs (P=0.015). Data are shown as mean ± SEM (PBL: n=4; Raw264.7: n=6) of percent DNA in tail. Student’s t-test: *P<0.05; **P<0.01; ***P<0.001.

Abbreviations: Au NP, gold nanoparticle; C+, positive control (10 μM hydrogen peroxide); C−, negative (untreated) control; DNA, deoxyribonucleic acid; EndoIII, endonuclease-III; Fpg, formamidopyrimidine-DNA glycosylase; h, hours; PBL, peripheral blood lymphocytes; SEM, standard error of the mean.

Table 4 Proliferation and genotoxicity evaluated by CBMN Cyt assay in Raw264.7 cells exposed to an absolute number of Au NPs

	CBPI	MN	NPB	NBUD
C−	1.810±0.017	2.3±0.3	1.0±0.4	0.8±0.4
C+	1.295±0.046***	28.7±2.4***	5.8±0.8**	6.2±0.6***
Au NP 5 nm
1×10^9	1.681±0.005**	4.3±0.7	1.0±0.4	0.8±0.4
1×10^10	1.673±0.013**	3.7±0.3*,a	1.3±0.4	1.2±0.2
1×10^11	1.661±0.014**	4.7±0.3**,a	1.5±0.3^a	1.0±0.4^a
Au NP 15 nm
1×10^9	1.678±0.005**	6.3±0.6**	1.3±0.2	1.3±0.2
1×10^10	1.672±0.011**	6.0±0.5**,b	2.0±0.4	1.8±0.4
1×10^11	1.665±0.021**	9.3±1.2**,b	2.3±0.2*,b	2.3±0.2*,b

Notes: CBPI, MN, NPB, and NBUD frequencies scored by CBMN Cyt assay after Raw264.7 cells exposed to absolute Au NPs numbers. MN frequency in Raw264.7 exposed to 15 nm Au NPs showed a dose-dependent relationship (P=0.008), as well as the NPB after exposure to 5 nm and 15 nm Au NPs (P=0.05 and P=0.02). Dose-dependent NBUD formation was scored in the presence of 15 nm Au NPs (P=0.008). Statistically significant differences from C− were determined by the Student’s t-test (*P<0.05; **P<0.01; ***P<0.001), as well as the differences between respective tested conditions (^aP<0.05 versus 15 nm Au NPs; ^bP<0.05 versus 5 nm Au NPs). Data represent the mean ± SEM (n=6).

Abbreviations: Au NP, gold nanoparticle; C+, positive control (0.1 μg/mL mitomycin C); C−, negative (untreated) control; CBMN Cyt, cytokinesis-block micronucleus cytome; MN, micronuclei; NBUD, nuclear buds; NPB, nucleoplasmic bridges; PBL, peripheral blood lymphocytes; SEM, standard error of the mean.

Figure 5 Cell viability in Raw264.7 after 2 hours and 24 hours of exposure to absolute numbers of 5 nm and 15 nm Au NPs.

Notes: Cells were exposed to 1.00×10⁹, 1.00×10¹⁰, and 1.00×10¹¹ Au NPs and evaluated by MTT assay. The data show the percent of living cells normalized to untreated control (±SEM, n=9). Statistically significant differences from the C− were determined by the Student’s t-test (*P<0.05). There were no statistically significant differences between the same number of 5 nm and 15 nm Au NPs.

Abbreviations: Au NP, gold nanoparticle; C+, positive control (10 μM hydrogen peroxide); C−, negative (untreated) control; h, hours; MTT, methylthiazol tetrazolium; SEM, standard error of the mean.

Figure S1 Cell viability evaluated by MTT assay in function of the theoretically calculated number of 5 nm and 15 nm Au NPs.

Notes: Mass concentrations (0.1–100 μg/mL) were expressed as theoretically calculated number of Au NPs ranging from 2.93×10¹² to 7.92×10¹⁶. In PBL (A and C) and Raw264.7 (B and D), after 2 hours (A and B) and 24 hours (C and D) treatment, no statistically significant differences among the cytotoxicity of 5 nm and 15 nm Au NPs were observed. Data show the percent of viable cells normalized to untreated control (± SEM, n=9). Statistical analysis was performed by Student’s t-test.

Abbreviations: Au NP, gold nanoparticles; MTT, methylthiazol tetrazolium; PBL, peripheral blood lymphocytes; SEM, standard error of the mean.

Figure S2 CBPI and MN frequency evaluated by CBMN Cyt assay in function of the theoretically calculated number of 5 nm and 15 nm Au NPs.

Notes: While CBPI shows no differences in antiproliferative activity of 5 nm and 15 Au NPs in PBL (A) and Raw264.7 (B), MN frequency was higher in PBL (C) and Raw264.7 (D) exposed to 15 nm Au NPs than to 5 nm (P<0.05). Data are shown as mean ± SEM (PBL: n=4; Raw264.7: n=6). Statistical analysis was performed by Student’s t-test.

Abbreviations: Au NP, gold nanoparticle; BN, binucleated cells; CBMN Cyt, cytokinesis-block micronucleus cytome; CBPI, cytokinesis block proliferation index; MN, micronuclei; PBL, peripheral blood lymphocytes; SEM, standard error of the mean.