Genotoxicity of aluminium oxide, iron oxide, and copper nanoparticles in mouse bone marrow cells

The Fe₂O₃ (4–8 nm), Al₂O₃ (40 nm), and Cu (40 nm) nanoparticles used in this study were purchased from Plasma Chem (Berlin, Germany) and characterised elsewhere (31). Briefly, the size and morphology of nanoparticles were observed with a transmission electron microscope (TEM) JEM-1400 (Jeol, Tokyo, Japan) at 80 kV and 40000x magnification. Hydrodynamic radius determined with a Zetasizer Nano ZS analyser (Malvern Instruments Ltd, Malvern, UK) in extensively sonicated water suspensions of nanoparticles (25–50 μg/mL) showed much higher average diameter of Fe₂O₃, Al₂O₃, and Cu nanoparticles than declared (16±5 nm, 59±8 nm, and 51±4 nm, respectively), most likely due to agglomeration in water (32).

Male BALB/c mice (6–7 week old) weighing ~22±11 g (n=135) were obtained from the National Institute for Biotechnology and Genetic Engineering (NIBGE, Punjab, Pakistan) and kept in plastic cages (2–3 per cage) with saw dust beddings in a well-ventilated room with natural light under controlled temperature (22±3 °C) and relative humidity (55 %+5 %). The mice had free access to food and water and were marked with different colours for identification. All animal experiments were approved by the NIBGE Animal Care and Use Committee.

Table 1 details the experimental design with groups treated with different nanoparticle doses and negative and positive controls. Nanoparticle doses were selected based on our preliminary dose-response experiments. With Al₂O₃ and Fe₂O₃ nanoparticles we observed no signs of toxicity, even at the highest tested concentration of 50 mg/kg body weight (bw), but with Cu nanoparticles we had to lower the dose to 15 mg/kg bw, as even at 20 mg/kg bw it caused muscle tremors, paralysis, increased heart rate, hypoventilation, and coma.

NC – negative control; PC – positive control; MMC – mitomycin C; MMS – methyl methanesulphonate

Experimental doses were obtained by further dissolving 10 mg/mL stock solutions with water followed by vortexing and sonication. Doses were administered in a volume of 20 mL/kg body weight intraperitoneally (ip) for 14 consecutive days. Intraperitoneal administration of drugs in suspension and/or nanoparticle formulations has been evidenced to result in faster and more complete absorption compared to oral and or subcutaneous routes. Furthermore, it is generally considered that systemic exposure to a substance given intraperitoneally is closer to that of the intravenous route (33).

A single ip dose of mitomycin C (2 mg/kg) was used as positive control in chromosome aberration (CA) and micronucleus (MN) assay. For the comet assay we used a single ip dose of methyl methanesulphonate (100 mg/kg) (both from Sigma-Aldrich, St. Louis, MO, USA) as positive control. Negative controls were injected with distilled water.

The experiment followed the protocol described elsewhere (34) with slight modifications. One hour and a half before sacrifice in a chamber filled with carbon dioxide (which occurred 24 h after the administration of the last nanoparticle dose), the mice received a single ip dose of 2 mg/kg colchicine (Sigma-Aldrich) to arrest cell division at metaphase and their femurs were removed. Bone-marrow cells were harvested from femurs, treated with 0.56 % KCl hypotonic solution (Sigma-Aldrich), and kept in a water bath at 37 °C for 25 min. Then they were centrifuged at 2000 g for 10 min and cell pellets immersed in ice-cold ethanol and acetic acid fixative (Fisher Scientific, Pittsburgh, PA, USA) (3:1, v/v) and washed five times at 20-min intervals. Cell pellets were then suspended in a small amount of fixative and a few drops placed on pre-cleaned and chilled microscope slides. The slides were air-dried for 3–5 min before staining with freshly prepared 5 % Giemsa stain (MP Biomedicals, Hutton, CA, USA).

The slides prepared for the CA assay were also used to calculate the mitotic index (MI) by counting mitotic cells at metaphase in 1000 cells per animal (totalling 5000 cells per treatment and control groups) with a light microscope (100x magnifying oil immersed lens, Nikon, Tokyo, Japan) and multiplying them by 100 to obtain percentage (35, 36).

Total CAs were counted in 2500 metaphases for each treatment and controls (500 per animal).

The MN assay followed the protocol described elsewhere (37, 38). Bone marrow cells were harvested using foetal calf serum (2 mL) 24 h after receipt of the last dose. Cell pellets obtained by centrifugation at 300 g for 5 min were then dissolved again in about 500 μL of foetal calf serum.

Two smears were prepared for each treatment and air-dried prior to fixing in 90 % methanol at -20 °C for 20 min and staining with acridine orange (MP Biomedicals) for 2 min. After washing with phosphate buffer (Invitrogen, Carlsbad, CA, USA) twice for 3 min each, two slides per dose group were coded and scored blindly for MN in about 1000 reticulocytes (RETs) or polychromatic erythrocytes (PCEs) per slide at 1000x magnification under UV light using an Olympus BX50 fluorescent microscope (Southend-On-Sea, UK). We also determined the percentage of RETs or PCEs/normochromatic erythrocytes (NCEs) per 1000 cells, as any reduction in the number of PCEs or RETs is a sign of bone marrow toxicity.

Bone marrow cells were harvested from femurs into a microcentrifuge tube containing 1 mL of cold Hank’s balanced salt solution (HBSS) (Thermo Fisher Scientific, Pittsburgh, PA, USA), 0.02 mol/L ethylenediaminetetraacetic acid (EDTA) (Gibco-BRL, Life Technologies Ltd., Inchinnan, UK), and 10 % dimethyl sulphoxide (DMSO) (Thermo Fisher Scientific). Bone marrow suspension was filtered with a 40 μm cell strainer into 15 mL conical tubes on ice. The alkaline comet assay followed the procedure described elsewhere (39, 40). Briefly, we prepared a mixture of single-cell suspension (100 μL) containing approximately 2×10⁶ cells/mL and 1 % low melting point agarose (LMA) (Promega Corporation, Madison, WI, USA) with 900 μL of phosphate buffer saline (Gibco-BRL) and spread 200 μL of the mixture over microscope slides precoated with 1 % normal melting point agarose (NMA) (Invitrogen Life Technologies Ltd., Paisley, UK) and then covered the slides with a cover slip. The slides were left to solidify at 4 °C for 30 min and then the cover slips were removed. Two slides were prepared for each sample.

Slides were immersed into a fresh cold lysis solution prepared at least one hour in advance of use and containing 2.5 mol/L NaCl (Sigma-Aldrich), 0.1 mol/L EDTA, 10 % DMSO, 1 % Triton X-100 (Applichem GmbH, Darmstadt, Germany), and 0.01 mol/L Tris-HCl (Merck, Whitehouse Station, NJ, USA) or NaOH (Sigma-Aldrich) to adjust it to pH 10. Following lysis, the slides were placed into a chilled alkaline solution (0.3 mol/L NaOH and 0.001 mol/L EDTA, pH >13) for 40 min to get DNA unwound. Then they were subjected to electrophoresis (in the same alkaline solution) at 0.8 V/cm, ~300 mA, and 4 °C in the dark for 30 min and neutralised to pH 7.5 with 0.4 mol/L Tris HCl three times for 5 min each. After fixing with ice cold ethanol (100 %) and staining with 20 μg/mL ethidium bromide (Sigma-Aldrich), the slides were left to dry overnight.

A total of 50 comets were scored visually at 40x magnification with an epifluorescence microscope (LB-201, Labomed Inc., Los Angeles, CA, USA) on each of the two slides per dose. Total score ranged between 0 (no detectable damage) and 400 (maximum damage) according to the method described by Collins (41), as follows: AUT=N0×0+N1×1+N2×2+N3×3+N4×4 \mathrm{AU}_{\mathrm{T}}=\mathrm{N}_{0} \times 0+\mathrm{N}_{1} \times 1+\mathrm{N}_{2} \times 2+\mathrm{N}_{3} \times 3+\mathrm{N}_{4} \times 4

where AU_T are arbitrary units and N₀, N₁, N₂, N₃, and N₄ are the number of cells scored in each group (0, 1, 2, 3, and 4, respectively). The results from three independent experiments were averaged to obtain AU_T for each treatment (42).

To detect oxidative damage to DNA bases we used the human 8-hydroxyguanine DNA-glycosylase (hOGG1) and endonuclease III (EndoIII) modified comet assay as described elsewhere (41). Briefly, the assay followed the same experimental steps as the standard comet assay, except that, following lysis, the slides were washed with enzyme buffer instead containing 0.04 mol/L N-(2-hydroxyethyl) piperazine-N’-2-ethanesulphonic acid (HEPES), 0.1 mol/L KCl, 5 mmol/L EDTA, 0.2 mg/mL bovine serum albumin (BSA) (Sigma-Aldrich), and KOH (Merck) to adjust pH to 8.0.

After washing, two slides from each dose group were treated with 200 μL of buffer (without enzyme as negative control), 200 μL of enzyme buffer containing 1.6 U/mLhOGG1 (1:1000), and 200 μL of enzyme buffer containing 10 U/mL Endo III (1:1000) (New England Biolabs Ltd., Hitchin, UK). The slides were then incubated at 37 °C for 45 min.

After enzyme treatment, the DNA unwinding, electrophoresis, neutralisation, staining, and scoring of damaged DNA were performed in the same way as described above for the standard comet assay. The slides without enzyme treatment (negative control) served to estimate the background level of DNA strand breaks (SB) (43, 44).

Statistical analysis was run on Minitab version 16 (Minitab Inc., State College, PA, USA). One-way analysis of variance (ANOVA) and Tukey’s range test were used to establish significant (P<0.05) differences between the control groups and treatment groups.