Effect of low-dose exposure of aluminium oxide nanoparticles in Swiss albino mice: Histopathological changes and oxidative damage

Abstract

Rapid growth in the use of aluminium oxide nanoparticles (Al₂O₃ NPs) in various fields such as medicine, pharmacy, cosmetic industries, and engineering creates concerns since the literature is replete with data regarding their toxicity in living organisms. The objective of the present study was to demonstrate the potential toxicological manifestations of repeated exposure to Al₂O₃ NP at low doses in vivo. In the present study, Al₂O₃ NP was orally administered at 15, 30 or 60 mg kg⁻¹ body weight for 5 days to Swiss albino male mice. A battery of well-defined assays was undertaken to evaluate aluminium (Al) bioaccumulation, haematological and histological changes, oxidative damage and genotoxicity. Physico-chemical characterisation demonstrated increases in hydrodynamic diameter along the concentration gradient of Al₂O₃ NP dispersed in MilliQ water. Brain, liver, spleen, kidney and testes showed high Al retention levels. Histopathological lesions were prominent in the brain and liver. Al₂O₃ NP treatment increased levels of lipid peroxidation and decreased glutathione content in the test organs at all dose levels. The enzyme activities of catalase and superoxide dismutase were also significantly altered. DNA damage quantified using the comet assay was markedly increased in all the soft organs studied. Anatomical abnormalities, redox imbalance and DNA damage were positively correlated with Al retention in the respective organs. Size, zeta potential and colloidal state might have contributed to the bio-physico-chemical interactions of the NPs in vivo and were responsible for the non-linear dose response. The overall data indicate that Al₂O₃ NP exposure may result in adverse health consequences, inclusive of but not limited to disturbed redox homeostasis, hepatocellular toxicity, neurodegeneration and DNA damage.

Keywords

Aluminium oxide nanoparticles,DNA damage hepatocellular histopathology neurodegeneration ROS

Introduction

Aluminium oxide nanoparticles (Al₂O₃ NPs) are one of the most abundantly used engineered NPs. Dielectric and abrasive properties of Al₂O₃ NP encourage their wide usage in various industries (Kaste and Rice, 2004; Schrand et al., 2010). Occupational as well as non-occupational exposure to the metal occurs through personal care products, bone substitutes, antacids, buffered aspirins, drug delivery systems and other medical preparations (Krewski et al., 2007; Sadiq et al., 2011). The Organization for Economic Co-operation and Development Steering Group for Test Guidelines categorized Al₂O₃ NPs as a high-priority group in 2007. In comparison with bulk materials, the nanometer-sized materials exhibit enhanced toxicity in biological systems when released into the environment (Donaldson et al., 2001). For the past few decades, the increasing exposure of humans to these NPs has produced possible health hazards (Sadiq et al., 2011). Apart from being extremely beneficial to industrial applications, the accumulation of Al₂O₃ NP in the environment leading to an increased rate of human exposure is a major contributor to their toxicological profile. As of 2010, the global annual production of Al₂O₃ NP was 18,500 tons. With the constant rise in production, it is estimated that it can exceed 100,000 tons in 2020 (Asztemborska, 2018).Thus, there is a need to assess their nano-safety and identify the factors that influence their associated hazards.

For the successful application of nanomaterials in biomedicine, it is essential to understand their fate and potential toxicity in vivo. Aluminium (Al) itself and its related compounds are reported to exhibit genotoxicity both in vitro and in vivo (Krewski et al., 2007). Balasubramanyam et al. (2009) and Prabhakar et al. (2011) reported the induction of genotoxicity and oxidative stress in Wistar rats following acute oral exposure of Al₂O₃ NP at high concentrations of 500, 1000 and 2000 mg kg⁻¹ body weight (bw). Subacute exposure of Al₂O₃ NPs (500 mg kg⁻¹ bw) was able to induce oxidative stress and histological responses in rats (Shrivastava et al., 2013). Chen et al. (2008) reported altered mitochondrial potential, redox imbalance and decreased tight junction protein expression in human brain micro vascular endothelial cells (1 µM–10 mM Al₂O₃ NP) in vitro. Al was also reported to induce neurodegeneration, and its toxicity results from free radical damage (Wu et al., 2012).

Al₂O₃ NP toxicity acts through multiple direct and indirect mechanisms, which include oxidative stress as a pivotal factor. However, NPs can cause cellular stress responses at lower doses and for longer periods of time (Bell et al., 2014). Data on low-dose exposure of Al₂O₃ NP on mammals are scarce. Genotoxicity and other complex outcomes of NP exposure are dependent on the physico-chemical properties such as size, surface chemistry, agglomeration and stability of the NP (Nel et al., 2006).

The present study was designed to elucidate the toxic effects of low-dose multiple exposures of Al₂O₃ NP in a mammalian system. Since exposure to Al₂O₃ NP largely occurs by the ingestion of antacids, aspirins and other medical preparations (Krewski et al., 2007), the oral route of exposure was selected for this study. Swiss albino mice were orally administered with various doses of Al₂O₃ NP. A battery of well-defined in vivo assays was undertaken to assess the cytotoxic and genotoxic effects of these NPs with respect to Al accumulation in different soft organs.

Materials and methods

Chemicals

Al₂O₃ NPs (CAS no. 1344-28-1), normal melting point agarose, low melting point agarose, disodium salt of ethylenediaminetetraacetic acid (EDTA), Tris buffer (CAS no. 77-86-1), ethidium bromide (EtBr; CAS no. 1239-45-8), Triton X-100 and Tris hydrochloride (HCl) were purchased from Sigma-Aldrich Co. (St. Louis, Missouri, USA). Essential buffers of sodium phosphate, hydrogen peroxide, trichloroacetic acid (TCA), 2-thiobarbituric acid (TBA; CAS no. 504-17-6), 5-sulfosalicylic acid, sodium citrate, hydrogen peroxide, 5,5-dithiobis-2-nitrobenzoic acid (CAS no. 69-78-3), 1-chloro-2,4-dinitrobenzene (CAS no. 97-00-7), reduced glutathione (GSH; CAS no. 70-18-8), pyrogallol, phosphate-buffered saline (PBS; Ca⁺⁺, Mg⁺⁺ free PBS), Roswell Park Memorial Institute medium (RPMI 1640) and diethylenetriaminepentaacetic acid were purchased from Hi Media, Mumbai, India. Giemsa (CAS no. 51811-82-6) was purchased from Merck, India.

Preparation and characterization of Al₂O₃ NP

Al₂O₃ NPs were purchased from Sigma-Aldrich Co. (USA). Physical characterization provided by the supplier was particle size (<50 nm by transmission electron microscopy (TEM) and surface area (>40 m² g⁻¹ by Brunauer Emmett Teller ; BET). Al₂O₃ NP stock dispersion was prepared by suspending the powder in filter sterilized MilliQ water and sonicated for 30 min by ultrasonic vibration at 130 W. Stock dispersion was serially diluted to prepare the final working concentrations. The working concentrations were subjected to sonication and vigorous vortexing as per requirement.

Hydrodynamic characterization of Al₂O₃ NPs at equivalent concentrations to the doses used for animal experiments (15–60 mM or approximately 1.5–6 mg mL⁻¹ or approximately 15–60 mg kg⁻¹ bw in 10 mL suspension) was done by dynamic light scattering (DLS) using a Malvern Zetasizer Nano-S (Malvern Instruments, UK). The average hydrodynamic diameter (nm), polydispersity index (PDI) and zeta potential (ZP; mV) were recorded by Zetasizer Ver. 7.10 software. PDI values depicted the extent of particle agglomeration and ZP indicated the net charge of the particles in suspension. Since NPs were dispered in double distilled water (viscosity 10034 mm²/s; refractive index 1.33) for all treatments, the same solvent was used for the DLS experiments.

Shape, size and morphology of Al₂O₃ NP (1 µg mL⁻¹) were analysed using TEM (JEM-2100 LaB6, 200 kV; JEOL, Peabody, Massachusetts, USA). Two microlitres of the sonicated suspension were drop casted on a copper grid (type B copper mesh 300) and dried followed by observation by TEM.

Animal care and treatments

Swiss albino mice (8–10 weeks old and weighing 25–30 g) were purchased and acclimatized for 1 week in the institutional animal house. They were randomly housed in cages with autoclaved paddy husk for bedding (five animals per cage) and maintained at a temperature of 22 ± 2°C temperature, humidity of 50–70% and 14 h:10 h light and dark cycle. The animals were fed with standard rodent pellets (consisting of crude protein and fibre) and drinking water ad libitum. The guidelines set by the Committee for the Purpose of Control and Supervision of Experimental Animals, India, and the ethical committee of the University of Calcutta were adopted for all experiments. All mice used in this study received proper care and handling in compliance with the CPCSEA guidelines and all experimental procedures were approved by the ethical committee of the University Animal Care Unit, University of Calcutta (Approval no. #885/ac/06/CPCSEA).

The animals were divided into four experimental groups (five mice per group) as follows:

Group 1 – Control group, animals were gavaged with 0.3 mL of MilliQ water for 5 consecutive days.

Groups 2 to 4 – animals were gavaged once a day for 5 consecutive days with various concentrations of Al₂O₃ NP dispersion 15, 30 and 60 mg kg⁻¹ bw in 10 mL suspension, respectively. The NPs were dispersed in a fixed volume of 0.3 mL of MilliQ water for all mice weighing 30 g each.

On the seventh day, bws were recorded, intracardial blood samples were collected separately in EDTA (1.6 mg EDTA/mL)-coated vials for haematological studies and in non-heparinized vials for serum chemistry. Animals were sacrificed by cervical dislocation. Thereafter, brain, liver, spleen, kidney and testes were quickly removed, weighed and processed for the different assays.

Organosomatic index or relative organ to body weight percentage for each organ was calculated (Chirumari and Reddy, 2007) using the following formula:

Organosomatic index = (Weight of the organ/Total body weight on day 7) × 100

Part of the tissue samples was fixed for histopathology, ICP-AES study, and the rest was stored in a −80°C freezer for further use.

Histopathology

For histopathological studies, tissue samples from brain, liver spleen and kidney were fixed in 4% paraformaldehyde and stored overnight at 4°C. Fixed tissue samples were embedded in paraffin blocks and sections (5 µm) were made on a microtome. The tissue sections were stained with haematoxylin and eosin (HE) for analysis under light microscope.

Metal estimation using ICP-AES

Tissue samples were digested in Aqua regia solution using the wet digestion method. The concentration of Al in the respective tissue samples was analysed using ICP-AES (ARCOS, Spectro, Germany). The metal concentrations were expressed as mg/kg bw.

Sperm head abnormality assay

Sperm head abnormality assay was performed according to Aduloju et al. (2008). The morphological anomaly in the sperm head was categorized as described by Wyrobek and Bruce (1975) with modifications. Sperm head morphological anomalies such as banana head, hook, and amorphous were recorded.

Oxidative stress response

Membrane lipid peroxidation (LPO): Extent of membrane LPO, characterized by increased malondialdehyde (MDA), was estimated. Each tissue sample was homogenized in physiological saline and centrifuged at 11200 g for 10 min at room temperature. The supernatant was mixed with TCA-TBA to measure MDA levels following the method of Buege and Aust (1978). MDA was calculated using a molar extinction coefficient of 1.55 M⁻¹ cm⁻¹ and expressed as nanomoles of MDA formed per gram wet weight of tissue.

Enzymatic antioxidants (CAT, SOD): Catalase (CAT) and superoxide dismutase (SOD) activity was determined according to the methods of Aebi (1984) and Marklund and Marklund (1974), respectively. Ten per cent (w/v) tissue homogenate in PBS was centrifuged at 10000 g at 4°C for 15 min. The supernatant was used to estimate CAT and SOD activities separately. SOD activity was expressed as unit SOD/mg protein and CAT activity was expressed as IU/mg protein.

Non-enzymatic antioxidant (GSH): Sample tissues were homogenized in 0.02 M EDTA and centrifuged at 11200 g at 4°C for 30 min. The level of reduced GSH content in tissue homogenates was estimated according to Sedlak and Lindsay (1968).

Protein content of tissue homogenates was estimated by Bradford’s method (1976).

Genotoxicity assessment

Tissue portions of target organs were minced in RPMI-1640, diluted with PBS and cells were processed for comet assay according to the methods of Singh et al. (1988) with minor modifications (Iswarya et al., 2015). The slides, after electrophoresis, were dipped in neutralizing buffer – 0.04 M Tris HCl (pH 7.5). Following neutralization for 10 min, the slides were stained with EtBr (2 µg mL⁻¹) and viewed under fluorescence microscope (Leica, Wetzlar, Germany; excitation filter 515–560 nm and barrier filter of 590 nm) equipped with a CCD camera. Evaluation of the images was done by auto-image analysis (Komet version 5.5; Andor Technology, Nottingham, UK). For each sample three slides were prepared at each dose per animal to minimize the chance of error. Extent of DNA damage was quantified by scoring the median values of comet parameter – tail DNA (%) and expressed as mean values for each concentration. For every concentration (i.e. 50 nuclei from each slide), 150 nuclei were studied.

Statistical analysis

All experiments were conducted thrice for error minimization. Values are expressed as mean ± standard deviation. Sigma Stats.3 software (SPSS Inc., Chicago, Illinois, USA) was used for performing a one-way analysis of variance (for comet assay, study of oxidative stress response and biochemical parameters). The value of p < 0.05 was considered to be statistically significant for all cases.

Results

Characterization of Al₂O₃ NP

The hydrodynamic size of Al₂O₃NPs in MilliQ water was found in the range of approximately 202–261 nm. The hydrodynamic diameter increased with increasing treatment concentrations. ZP values ranged from approximately −31 to −25 mV at pH 7. The PDIs for the tested concentrations were < 0.2 (Table 1). TEM photomicrograph (Figure 1) depicts the particles to be spherical in outline with smooth surfaces and having a size range of approximately 10–55 nm. The particles were well distributed in all the observed fields with few loose clusters. Figure 2 shows the DLS size distribution and ZP curves. The highest concentration showed a decline in colloidal stability as displayed by the presence of NP populations occurring in two peaks with different sizes and least ZP value of approximately −25 mV.

Table 1.

Physico-chemical characterization of Al₂O₃ NP.^a

Concentration (mM)	Mean hydrodynamic diameter (nm; mean ± SD)	Poly dispersity index	ZP (mV)
15	202.5 ± 65.68	0.150	−31.8 ± 4.47
30	217.3 ± 85.75	0.164	−27.4 ± 5.05
60	261.5 ± 162.07	0.134	−25.8 ± 5.05

Al₂O₃ NP: aluminium oxide nanoparticle; SD: standard deviation; ZP: zeta potential.

^a Values are mean of three replicates ± SD.

Organosomatic index

Oral gavage of Al₂O₃ NP caused no significant change in bw. The organosomatic indices for all the tested organs (except for the kidney in mice exposed to 60 mg kg⁻¹ bw of Al₂O₃ NP) showed no significant change with respect to that of control (Figure 3).

Haematology and serum chemistry

The haematological parameters did not show any significant change except for the rise in the number of lymphocytes in mice exposed to highest concentration of Al₂O₃ NP (60 mg kg⁻¹ bw; Table 2). The aspartate aminotransferase (AST) and alanine aminotransferase (ALT) values increased significantly at 30 and 60 mg kg⁻¹ bw of Al₂O₃ NP (Table 3). The other parameters did not show any significant changes compared to the respective controls.

Table 2.

Haematological parameters of mice orally gavaged with various concentrations of Al₂O₃ NP (15, 30 and 60 mg kg⁻¹ bw) for 5 consecutive days.^a

Parameter	Unit	Control	15 mg kg⁻¹ bw	30 mg kg⁻¹ bw	60 mg kg⁻¹ bw
WBC	10⁹ (L)	6.9 ± 1.40	6.7 ± 1.58	6.2 ± 0.87	5.5 ± 1.08
Neutrophil	%	34.24 ± 3.48	33.04 ± 3.12	30.0 ±3.01	25.0 ± 1.95
Lymphocyte	%	58.60 ± 0.45	60.18 ± 5.28	63.0 ± 2.92	70.0 ± 3.13^b
Monocyte	%	3.18 ± 0.27	3.30 ± 0.95	4.0 ± 0.07	4.0 ± 0.05
Eosinophil	%	2.0 ± 0.08	1.60 ± 0.01	1.0 ± 0.02	1.0 ± 0.01
Basophil	%	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0
RBC	10¹² (L)	7.68 ± 1.45	7.88 ± 2.04	8.31 ± 0.95	8.57 ± 0.72
Hb	g dL⁻ ¹	10.5 ± 2.64	10.5 ± 2.23	10.8 ± 1.07	11.9 ± 0.98

Al₂O₃ NP: aluminium oxide nanoparticle; SD: standard deviation; WBC: white blood cells; RBC: red blood cells; Hb: haemoglobin; bw: body weight.

^a Data are mean ± SD of five mice per group.

^b Significant from control at p < 0.05.

Table 3.

Serum chemistry of mice orally gavaged with various concentrations of Al₂O₃ NP (15, 30 and 60 mg kg⁻¹ bw) for 5 consecutive days (N = five mice per group).^a

Parameter	Unit	Control	15 mg kg⁻¹ bw	30 mg kg⁻¹ bw	60 mg kg⁻¹ bw
TP	g dL⁻ ¹	5.8 ± 0.82	7.1 ± 0.41	7.0 ± 1.01	6.0 ± 1.08
TB	mg dL⁻ ¹	0.5 ± 0.01	0.2 ± 0.001	0.4 ± 0.01	0.3 ± 0.01
ALB	g dL⁻ ¹	2.9 ± 0.40	2.8 ± 0.08	2.9 ± 0.09	2.6 ± 1.95
AST	U L⁻ ¹	301.0 ± 21.93	402.0 ± 14.49	810.0 ± 18.95^b	750.0 ± 27.90^b
ALT	U L⁻ ¹	152.0 ± 12.25	172.0 ± 10.78	272.0 ± 13.10^b	245.0 ± 17.31^b
ALP	U L⁻ ¹	165.0 ± 13.92	127.0 ± 12.08	480.0 ±17.40^b	44.0 ± 2.01
Creatinine	mg dL⁻ ¹	0.8 ± 0.09	0.6 ± 0.05	0.5 ± 0.02	0.6 ± 0.09
Urea	mg dL⁻ ¹	47.0 ± 0.95	44.0 ± 2.58	42.0 ± 1.95	40.0 ± 2.72
Sugar	mg dL⁻ ¹	133.0 ± 7.0	128.0 ± 6.39	120.0 ± 4.31	112.0 ± 13.98
Potassium	mEq L⁻ ¹	7.0 ± 1.38	6.4 ± 2.01	6.8 ± 2.22	5.6 ± 1.23
Calcium	mg dL⁻ ¹	9.0 ± 2.10	7.8 ± 1.36	8.01 ± 2.06	6.7 ± 0.97
Phosphorous	mg dL⁻ ¹	4.2 ± 1.01	5.0 ± 0.89	8.0 ± 1.58	8.5 ± 1.74

Al₂O₃ NP: aluminium oxide nanoparticle; TP: total protein; TB: total bilirubin; ALB: albumin; AST: aspartate aminotransferase; ALT: alanine aminotransferase; ALP: alkaline phosphatase; bw: body weight; SD: standard deviation.

^a Values are mean ± SD of five mice per group.

^b Significant from control at p < 0.05.

Estimation of metal accumulation by ICP-AES

Figure 4 represents the accumulation of Al in brain, liver, spleen, kidney and testes. The highest accumulation was recorded in brain, liver and testes of mice exposed to 30 mg kg⁻¹ bw of Al₂O₃ NP. Al retention in brain at 30 mg kg⁻¹ bw was approximately 3 and 2.2 times greater than that at 15 and 60 mg kg⁻¹ bw of Al₂O₃ NP. In hepatic tissue, Al accumulation was 21 and 7 times greater in 30 and 60 mg kg⁻¹ bw of Al₂O₃ NP, respectively, in comparison to that at 15 mg kg⁻¹ bw of Al₂O₃ NP. In testes, Al burden was significantly increased (p < 0.05) in those treated with 30 and 60 mg kg⁻¹ bw of Al₂O₃ NP in comparison to that treated with 15 mg kg⁻¹ bw of Al₂O₃ NP. Dose-dependent increase in Al accumulation was observed in spleen and kidney.

Histopathology

Histological abnormalities were observed in HE stained microtome sections of brain and liver tissues of mice gavaged with 30 and 60 mg kg⁻¹ bw of Al₂O₃ NP. Neurofibrillary tangles were observed in brain cortical sections. Liver sections demonstrated dilated central vein (DCV) and expanded portal tract (EPT). No gross anatomical lesion was noted in spleen and kidney sections (Figure 5(a)).

Sperm head abnormality assay

All morphological anomalies (including banana head, pin head, hook less, amorphous) were classified into one category in order to evaluate sperm head abnormality as a single endpoint (Figure 5(b) and (c)). Abnormalities in sperm head morphology were significant (p < 0.05) in mice exposed to 30 and 60 mg kg⁻¹ bw of Al₂O₃ NP.

Oxidative stress response

Generation of MDA is one of the most reliable markers for estimation of the extent of membrane LPO. MDA level was observed to be maximum in brain, liver and testes of mice administered 30 mg kg⁻¹ bw Al₂O₃ NP. Significantly high (p < 0.05) MDA levels were also observed in liver and kidney of mice exposed to the highest concentration of Al₂O₃ NP (60 mg kg⁻¹ bw; Figure 6(a)). Increased CAT activity was observed in liver, spleen and kidney, whereas SOD activity decreased at all doses in the organs studied. CAT activity was significantly decreased in comparison to that of control in all treatment dose groups in testes and brain. In spleen, brain and kidney, significant (p < 0.05) inhibition of SOD activity was observed in mice gavaged with 30 mg kg⁻¹ bw of Al₂O₃ NP, while a similar effect was observed in liver and testes at Al₂O₃ NP (60 mg kg⁻¹ bw; Figure 6(b) and (c)). GSH level was inhibited at all tested doses of Al₂O₃ NP. Level of reduced GSH at 30 mg kg⁻¹ bw was significantly (p < 0.05) decreased in brain, liver and kidney, and at 60 mg significantly decreased levels of GSH were observed in liver, kidney and testes (Figure 6(d)).

Genotoxicity assessment

DNA strand break, characterized as % tail DNA, in brain, testes and spleen was significantly increased (p < 0.05) in mice at all tested concentrations of Al₂O₃ NP (Figure 7). Among the organs studied, the greatest DNA damage was noted in brains of mice exposed to 15 mg kg⁻¹ bw. The extent of DNA damage was greatest in brain followed by liver, spleen and kidney at 30 mg kg⁻¹ bw of Al₂O₃ NP exposure. Kidney and spleen showed a dose-dependent increase in % tail DNA.

Discussion

The growing commercial applications have brought Al₂O₃ NP under toxicologists’ purview. The present report deals with the toxicity assessment of low dose, repeated exposure of Al₂O₃ NP in Swiss albino male mice. The large surface area and extremely small size of the NP render it easily penetrable into the system through oral, dermal and nasal routes. In this multi-endpoint study, the oral route of exposure was adopted as it is a chief constituent of common antacids and other medical preparations.

Average particle diameter, size distribution and charge affect the in vivo and in vitro activities of the NPs. In our study, the TEM photomicrograph shows that the NPs were spherical and uniformly dispersed with few loose aggregates. The primary size was determined to be within the range of 10–55 nm, which was approximately within the range of <50 nm as provided by the manufacturer. Measurements of ZP allow for predictions – about the hydrophobicity, stability and indirect measure of surface charge of colloidal dispersion. Higher ZP values of Al₂O₃ NP are indicative of the fact that the particles are stable in the suspension. The PDI data indicated values <0.5, which indicated the onset of polydispersity in suspension. The values obtained from DLS are usually greater than those measured by other techniques such as TEM/AFM that indicate primary size. In our study, we found the hydrodynamic size of the particles to be greater than the primary size and it increased with increasing concentration. A much lower concentration was used for primary characterization for distinct visualization of each particle. The deviation of hydrodynamic diameter from the primary size could also be due to the possible presence of a hydronium ion shell around the NPs in suspension (Jiang et al., 2009). Moreover, the increase in concentration of NP in the dispersion might have led to the formation of a network of repulsive particles and the repulsive interaction tends to centre the particles in a cage formed with adjacent suspended particles. This in turn changes the self-diffusion and thereby increases the hydrodynamic diameter (Monterio et al., 1999).

Organosomatic index reflects overall physical status of an organism. Body weight, food intake, and organosomatic index are simple biomarkers of toxicity (Das and Gupta, 2014). The overall physical status of experimental animals and their haematological profiles did not reflect any signs of acute toxicity.

As evident from ICP-AES studies, a significant amount of Al accumulated in all the test organs. Recent studies suggest that NPs can easily penetrate the biological barriers like GI barriers, the blood–brain barrier (BBB) and blood testes barrier in living systems (Kim et al., 2006). Following oral exposure, only 1% of ingested bioavailable forms of Al (aluminium citrate/NPs) get absorbed into the system (Balasubramanyam et al., 2009). Significant tissue distribution of Al was observed in brain, liver, kidney, spleen and testes of mice exposed to all concentrations of Al₂O₃ NP. The greater accumulation of Al at the 30 mg kg⁻¹ bw dose than that of the 60 mg kg⁻¹ bw dose in brain and liver can be correlated with the increase in mean hydrodynamic diameter with increasing concentration of Al₂O₃ NP. At high concentrations, the increase in particle size might have led to agglomeration that hindered their uptake. This suggests that size of NPs plays an important role in its accumulation in different tissues. Our results are suggestive of the fact that Al₂O₃ NPs could cross the GI barriers and enter into the circulating blood. This transport procedure is primarily accomplished through passive diffusion by binding to certain ligands in the blood and delivered to various organs via receptor mediated endocytosis, phagocytosis and pinocytosis (Di Virgilio et al., 2010; Simon-Deckers et al., 2008).

Liver belongs to the reticuloendothelial system where maximum absorption and storage takes place through liver sinusoidal endothelial cells and macrophage actions of the hepatocytes (Puntarulo, 2005). The nanosized material gets absorbed in blood through the enterocytes (Simon-Deckers et al., 2008). The changes in liver function test parameters – alkaline phosphatase (ALP), AST and ALT showed significant changes in mice administered with 30 mg kg⁻¹ bw of Al₂O₃ NP to that of control. ALP is a prominent marker of any imbalance in normal drainage of the biliary tree. Increased AST and ALT is indicative of impaired functions of hepatocytes. Histological abnormalities like DCV and EPT were profound in hepatic tissues of mice treated with 30 and 60 mg kg⁻¹ bw of Al₂O₃ NP but were absent at the lowest concentration (15 mg kg⁻¹ bw).

Infiltration of metal was increased in brain tissue as observed in the ICP-AES study. A significant amount of Al was detected in brain tissues of mice treated at all test concentrations of Al₂O₃. Chen et al. (2008) reported nano-alumina causing decreased expression of tight junction proteins like claudin-5 and occludin thus negatively affecting BBB permeability. In our experiment, Al₂O₃ NP exposure might have caused hyper permeability of BBB in mice. The circumventricular organs, which are not protected by the BBB, might have played a vital role in the deposition of Al in the cerebral tissue (Kim et al., 2006). In the present study, the hydrodynamic sizes of NPs were in the submicron range (100–1000 nm). Thus, Al could not have entered in the brain tissue through the intact BBB because BBB allows particles <100 nm to pass through it via transcytosis. Detectable changes in brain morphology were noted in mice administered with 30 and 60 mg kg⁻¹ bw of Al₂O₃ NP. The neurofibrillary pathology, which is commonly associated with neurodegenerative disease, was observed in our study. Thus, the high amount of Al accumulation in brain also conforms to the role of Al in causing neurodegeneration.

Normal spleen anatomy shows two morphologically distinct regions – the red pulp and the white pulp. In the mouse, extramedullary haematopoiesis occurs in the red pulp. The red pulp macrophages are actively phagocytic and remove blood-borne particulate matter. This corroborates with Al burdens in the spleen at all tested concentrations with the greatest accumulation in mice administered with 60 mg kg⁻¹ bw of Al₂O₃ NP. Mild degeneration of tissue was observed in mice exposed to 30 and 60 mg kg⁻¹ bw of Al₂O₃ NP. Renal tubular damage and glomerular sclerosis were reported in rats exposed to Al stress (Contini et al., 2015). In our study, Al₂O₃ NP exposure and subsequent Al bioaccumulation did not induce any such anomalies in renal histology. There was also no significant change in kidney function tests with respect to control. The amount of Al was thus not enough to produce any histopathological changes in both spleen and kidney. High amounts of Al accumulated in testes of mice as NPs can successfully cross the blood testes barrier (Kim et al., 2006). Greater levels of abnormalities in sperm morphology were observed in animals gavaged with 30 mg kg⁻¹ bw of Al₂O₃ NP. Although maximum uptake was observed at 60 mg kg⁻¹ bw in the testes, it is possible that the NPs did not internalize within the sperm cells due to possible agglomeration. Hence, in a future study, internalization within sperm cells could be qualitatively studied by various microscopy techniques and quantitatively by ICP-AES to support this hypothesis. A study by Długaszek et al. (2000) also showed varying concentrations of Al in tissues of mice gavaged with Al. Significant changes in mouse sperm parameters due to exposure to iron oxide NPs were also reported (Varzeghani et al., 2018). Moreover, Al₂O₃ NP has been reported to induce spermatotoxicity (Yousef et al., 2016) and neuro-developmental toxicity (Zhang et al., 2018) in mice. The effect of low doses of Al₂O₃ NP on developmental toxicity and spermatogenesis are areas for further investigation.

The nano-sized objects after successful entry into the organs are known to induce prominent cytotoxic, genotoxic, oxidative stress and inflammatory responses (Dey et al., 2008; Hussain et al., 2005; Jeng and Swanson, 2006). In our study, increased cellular oxidative stress was recorded in liver, kidney, brain, spleen and testes of animals. Similar trends in oxidative stress responses in mice treated with Al₂O₃ NP at 500 mg kg⁻¹ bw and higher doses were reported by Prabhakar et al. (2011). Membrane LPO is among the most prominent markers of free radical damage. It is known that upregulation of CAT and downregulation of SOD activity are attributed to increased flux of H₂O₂ (Fernandez-Urrusuno et al., 1997; Powers and Jackson, 2008). The increased redox imbalance observed in all the test organs is consistent with metal accumulation at doses 30 and 60 mg kg⁻¹ bw. SOD is among the first line of defences against adverse effects of oxy radicals (Li et al., 2010). Excess utilization of GSH in neutralizing the free radicals is one of the probable reasons for the decrease in its level. Increased LPO depleted GSH, and lowered SOD and CAT activity is in agreement with the DNA damage data. Mice administered 30 and 60 mg kg⁻¹ bw of Al₂O₃ NP demonstrated greater amounts of DNA damage in terms of increased percentages of tail DNA. The percentage of tail DNA in brain and liver was increased at all tested concentrations of Al₂O₃ NP. Increased DNA damage recorded in spleen tissues of mice at all concentrations could be correlated with the increased levels of Al retention in the tissues. Membrane LPO and CAT activity increased in a dose-dependent manner while that of SOD and GSH decreased in comparison to that of control in the kidneys. Testes also reflected a state of increased oxidative burden, which is consistent with the DNA damage results. In general, oxidative stress and DNA damage were evident in all the test organs in mice exposed to 30 and 60 mg kg⁻¹ bw Al₂O₃ NP. Specifically, 30 mg kg⁻¹ bw incited higher toxicological responses than 60 mg kg⁻¹ bw presumably because of higher agglomeration at 60 mg kg⁻¹ bw that might be the underlying cause of lower internalization. Moreover, the possibility that the suspensions were diluted due to water consumption ad libitum over a period of time cannot be ruled out. This can in turn alter the observed hydrodynamic characteristics as a function of time and serial dilution following water intake. Conversely, following oral gavage, the NPs are taken up by the organs of the reticuloendothelial system and move through the blood stream into distant organs such as the brain. The possibility that the animals consumed a large volume of water right after oral gavage leading to immediate dilution of the NP suspensions is ambiguous and has not been explored as an objective of this study. This can open up future prospects in the study of pharmacokinetics and pharmacodynamics of Al₂O₃ NPs in vivo. However, further studies involving characterization of the NPs at each dose and correlating the results with microscopic visualization of the internalized particles might throw light on this speculation. With the increased release of Al₂O₃ NP into the environment, results of the present study can be extrapolated to predict their toxicological consequences in human systems.

Conclusion

Results of the current study confirmed the toxicity of Al₂O₃ NP in mice that was related to their physico-chemical properties like size, surface charge and stability. The histopathological alterations observed in brain, liver and spleen were related to the accumulation of NPs in these organs. A maximum Al₂O₃ NP retention and damage to brain, liver and testes was noted at the dose of 30 mg kg⁻¹ bw. Oxidative stress and DNA damage was manifested in the brain, liver, kidney, spleen and testes of mice that could be correlated with Al accumulation in these organs. The data obtained from this multi-parametric study supports the scientific knowledge in predicting the behaviour of engineered nanomaterials in the body.

Supplemental material

Supplementary_File-converted - Effect of low-dose exposure of aluminium oxide nanoparticles in Swiss albino mice: Histopathological changes and oxidative damage

Supplementary_File-converted for Effect of low-dose exposure of aluminium oxide nanoparticles in Swiss albino mice: Histopathological changes and oxidative damage by Arpita De, Swarupa Ghosh, Manoswini Chakrabarti, Ilika Ghosh, Ritesh Banerjee and Anita Mukherjee in Toxicology and Industrial Health

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Anita Mukherjee

Supplemental material

Supplemental material for this article is available online.

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Supplementary Material

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Effect of low-dose exposure of aluminium oxide nanoparticles in Swiss albino mice: Histopathological changes and oxidative damage

Abstract

Keywords

Introduction

Materials and methods

Chemicals

Preparation and characterization of Al2O3 NP

Animal care and treatments

Histopathology

Metal estimation using ICP-AES

Sperm head abnormality assay

Oxidative stress response

Genotoxicity assessment

Statistical analysis

Results

Characterization of Al2O3 NP

Organosomatic index

Haematology and serum chemistry

Estimation of metal accumulation by ICP-AES

Histopathology

Sperm head abnormality assay

Oxidative stress response

Genotoxicity assessment

Discussion

Conclusion

Supplemental material

Supplementary_File-converted - Effect of low-dose exposure of aluminium oxide nanoparticles in Swiss albino mice: Histopathological changes and oxidative damage

Footnotes

Declaration of conflicting interests

Funding

ORCID iD

Supplemental material

References

Supplementary Material

Preparation and characterization of Al₂O₃ NP

Characterization of Al₂O₃ NP