**1. Introduction**

Alumina nanoparticles (Al2O3 NPs) are among the most widely used and produced particles in the world, with a wide range of interesting applications such as biosensors, desalination, high-risk pollutants detection, capacitors, solar cell devices, and photonic crystals [1]. These NPs can also be retrieved as pollutants in the environment.

Al2O3 NPs are produced in huge quantities as by-products of water treatment (water treatment residuals), bauxite processing (red mud), and hard and brown coal burning in power plants (fly ash) [2]. Welding fumes and propellant combustion residues also seem to be the main sources of Al2O3 NPs occupational exposures [3,4]. Nanoparticle forms are particularly studied in toxicological research because of their increasing use and the concerns they raise. Indeed, unique physico-chemical properties of NPs such as small size (<100 nm) and high specific surface area, confer high surface reactivity and uncertainty toward their potential toxicity [5]. Workers' exposure to Al2O3 NPs can mainly occur by inhalation. Occupational exposures to these dusts are described in the literature to have deleterious health effects on the respiratory and nervous systems [3,6–9]. Therefore, it is necessary to improve understanding of NPs toxicity in order to redesign strategies to mitigate/reduce environmental and/or health impact [10]. Due to the lack of studies, research efforts are needed to better explore and thus protect predisposed populations such as workers.

This review focuses on biological effects described on the pulmonary system and associated described pathologies. Clinical, animal, and in vitro studies are successively presented in this paper. Briefly, workers exposed to Al2O3 NPs developed pneumoconiosis, also named "aluminosis". They also presented local respiratory symptoms such as cough or shortness of breath, inflammation in the lung parenchyma, probably long-term

**Citation:** Dekali, S.; Bourgois, A.; François, S. Critical Review on Toxicological Mechanisms Triggered by Inhalation of Alumina Nanoparticles on to the Lungs. *Biomedicines* **2022**, *10*, 2664. https://doi.org/10.3390/ biomedicines10102664

Academic Editor: Alberto Ricci

Received: 30 September 2022 Accepted: 19 October 2022 Published: 21 October 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

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pulmonary fibrosis, and increased risk of developing lung cancer [3,8,9,11]. Pulmonary aluminosis is a rare form of pneumoconiosis caused by aluminum or alumina powders [12,13]. Consequently, the majority of animal studies have focused on pro-inflammatory mechanisms triggered by the inhalation of Al2O3 NPs [14,15]. Results showed increases in total protein, neutrophils, lymphocytes, and lactate dehydrogenase (LDH) concentration in broncho-alveolar lavages (BALF), corroborating pro-inflammatory effects of Al2O3 NPs and suggesting potential permeabilization of the alveolo-capillary barrier. Moreover, various pro-inflammatory cytokines were secreted in animal BALFs: TNFα, IL-6, IL-1β, IL-8, MIP-2, and IL-33 [14–16]. However, to the best of our knowledge, inflammatory mechanisms are poorly studied in vitro. This review also critically presents contradictory results on the cytotoxic and genotoxic effects of Al2O3 NPs on lung cells.

### **2. Clinical Studies on Health Effects after Inhalation of Alumina Particles**

A recent study was conducted on fifteen male students aged (mean, range) 24, 19–31 with normal lung function in an inhalation chamber for human exposure to atmospheric particulate matter (PM) [9]. The aim was to examine the effects of short-term exposure to Al2O3 particles (3.2 μm, crystallinity unknown) on inflammatory markers in induced sputum in healthy volunteers. Controlled inhalations with exposure levels commonly seen in primary aluminum production were used (below toxicological reference values, i.e., 2 h at 3.9 mg/m3). Authors showed 24 h after exposure increases in polymorphonuclear neutrophils (PNNs), total proteins, and IL-8 concentrations in the sputum, suggesting a marked pulmonary inflammation. Moreover, microarray analyses of mRNA abundance collected from sputum macrophages and pathway analysis showed changes in the expression of 46 genes identified in three major biological process groups: cell–cell signaling, gene expression, RNA damage and repair, and regulation of connective tissue assembly. Only localized respiratory effects were reported in this study, and no systemic effects were observed.

A prevalence of pulmonary fibrosis 300 times higher than that observed in the general population was evaluated in workers exposed to alumina for 25 years [17]. However, these results are questionable because of the exposure of subjects to other particulate compounds that may have contributed to the onset of these respiratory pathologies. Indeed, significant quantities of asbestos fibers have also been found in the lungs of some subjects who have developed pulmonary fibrosis. Workers' exposures to fumes from aluminum welding, which may contain alumina, has also led to the appearance of pneumoconiosis and pulmonary fibrosis in some cases [3,11]. A higher frequency of respiratory diseases, such as chronic obstructive pulmonary disease (COPD), has been observed in aluminum welders. In addition, decreases in lung function (forced expiratory volume in a second (FEV1)) have been observed in workers in the aluminum industry exposed to dust containing mainly alumina [18]. Another study evaluated the lung tissue of fourteen workers exposed to hard metals and aluminum oxide [19]. Among them, five workers underwent transbronchial biopsy showing diffuse interstitial inflammatory changes: two of them were asymptomatic, one had clinically evident disease with severe giant cell inflammation, and two other workers showed local inflammation.

Although some studies on alumina exposure have concluded the induction of respiratory pathologies and a decrease in lung function in humans, other studies have revealed contradictory results. Despite respiratory symptoms (cough, shortness of breath) observed in workers (preparers of powders for propulsion systems using duralium) exposed to aluminum and alumina NPs, no link between these compounds and the appearance of diseases respiratory problems could be demonstrated [8]. Similarly, the follow-up of a cohort of 521 men working in the production of abrasives (exposure to alumina particles, silicon carbides, and formaldehyde) 72 over 25 years did not show a significant increase in total or cancer mortality or the incidence of non-malignant respiratory disease [20]. Finally, monitoring of the pulmonary function of welders using aluminum did not show any deleterious effect, although respiratory symptoms were observed in these workers [21].

A summary of the results of clinical studies performed on health effects after inhalation exposure to alumina particles is available in Table 1.



**Table 1.** Summary of clinical studies on health effects after inhalation of alumina particles.

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### **3. Animal Studies of Pulmonary Biological Effects Triggered by Alumina Nanoparticles**

In order to study the pulmonary toxicity of particles, different animal exposure methods can be implemented. Inhalation exposure is the most physiological method. However, this is expensive, and it requires extensive technical skills to set up the exposure system, the reproducibility of aerosol generation, and to characterize the physico-chemistry of generated aerosols. Another commonly used method to study in vivo lung toxicity of particles is intratracheal instillation (IT). This technique allows administrating of precise and known doses of particle suspensions [22]. IT is less constrained than inhalation but not representative of environmental exposure conditions. It is also possible to use other methods of exposure, such as nasal instillation, IT aspiration, oropharyngeal aspiration, or oral exposure. These techniques are less physiological and rarely used in literature.

Although possessing important anatomical and physiological similarities with humans, large mammals such as monkeys and pigs are very rarely used for lung toxicity studies due to ethical aspects and their cost. The rat is the alternative recommended by Organisation for Economic Co-operation and Development (OECD) because it is a more qualified model, easier to implement, and remains representative of the human respiratory system [23–25]. This animal model is preferred to the mouse model because it has more anatomical similarities with humans than with mice [26]. This animal model is the most commonly used to study the pulmonary toxicity of alumina particles. These studies are presented considering the kind of animal exposure, i.e., inhalation or IT/intranasal exposures.

### *3.1. Nose-Only and Whole-Body Inhalation Exposures*

Kim and colleagues reported pro-inflammatory effects of alumina nanoparticles after repeated nose-only inhalation on Sprague-Dawley rats [15]. After 28 days of exposure (5 days/week) to Al2O3 NPs concentrations ranged between 0.2 and 5 mg/m<sup>3</sup> (size 11.94 nm; unknown crystallinity), they showed increases in the total number of cells, neutrophils, lymphocytes, LDH, TNFα, IL-6 in BALF. Moreover, they reported histopathological lesions with alveolar macrophage accumulation in four and eight cases of the 5 mg/m<sup>3</sup> group during exposure and recovery, respectively. Pro-inflammatory effects significantly decreased after 28 days of exposure, but neutrophils and LDH concentrations remained significantly elevated compared to control groups. Results obtained in this study demonstrated a strong inflammatory potential of these NPs when inhaled, and authors suggested a no-observed-adverse-effect level of 1 mg/m<sup>3</sup> concentration. Recently, Wistar rats were also nose-only exposed to a high concentration of Al2O3 NPs (size 13 nm; γ/δ crystallinity; 20.0–22.1 mg/m<sup>3</sup> aerosol) following two exposure scenarios: single (4 h) or repeated exposures (4 h/day for 4 days) [14]. After repeated exposures, total proteins and LDH concentrations in BALF were significantly increased, suggesting that the alveolo-capillary barrier was damaged. Additionally, a marked pro-inflammatory reaction was observed with increased concentrations of neutrophils, macrophages, IL-1β, TNFα, GRO/KC, and MIP-2. Moreover, another study showed that after 7 days of inhalation exposure of mice to Al2O3 NPs (size 40 nm; unknown crystallinity; dose of 0.4 mg/m3) in whole-body chamber emphysema and small airway remodeling in lungs can occur, accompanied by enhanced inflammation and apoptosis [16]. Authors demonstrated that protein tyrosine phosphatase, non-receptor type 6 (PTPN6), was down-regulated and Signal Transducer and Activator of Transcription 3 (STAT3) phosphorylated in response to Al2O3 NPs exposure, culminating in increased expression of the apoptotic marker Programmed cell death protein 4 (PDCD4). Moreover, IL-6 and IL-33 concentrations were significantly increased in BALF. Therefore, a decrease in PTPN6 may have deleterious effects at the molecular, cellular, and tissue levels, leading to the initiation of inflammation and apoptosis, ultimately resulting in the development of COPD-like lesions.

### *3.2. Intratracheal or Intranasal Exposures*

IT or nasal instillation are the best alternatives to inhalation exposures for pulmonary toxicity studies in animals. These techniques notably offer the possibility of exposing animals in a less costly way, with good control of the dose administered in bolus and technically less complex than exposure by inhalation. In Sprague-Dawley rats exposed by IT to 40 mg of Al2O3 particles (size 4.37 μm, γ/α crystallinity), an increase in the number of cells, mainly macrophages but also neutrophils, and fibronectin concentrations were shown in BALF [27]. These concentrations remain increased twelve months after instillation, suggesting a persistence of the inflammatory phenomenon induced by Al2O3 NPs. These results are consistent with those obtained by inhalation but provide information on the persistence over time of the observed acute effects. BALF analysis of Wistar rats exposed to Al2O3 NPs (6.3 nm, crystallinity unknown, 0.5 mL at 300 cm2/mL) by IT also induced acute pulmonary inflammation [7]. Increases in polymorphonuclear cells were measured in BALF 24 h after exposure. In addition, Al2O3 NPs showed hemolytic potential. The effects observed in this study seem to be correlated with the surface properties of Al2O3 NPs (surface charge represented by the zeta potential in particular). Indeed, a correlation between the zeta potential and the influx of granulocytes or the hemolytic power has been observed for NPs with a high zeta potential [7].

Exposure by nasal instillation of Sprague-Dawley rats demonstrated other deleterious effects of alumina NPs (size and crystallinity unknown, 1–40 mg/kg) [28]. A slight dose-dependent inflammation was observed, but impairment of alveolo-capillary barrier permeability was also revealed by the increase in total protein concentration in BALF. The fibrotic potential of Al2O3 particles was assessed in Sprague-Dawley rats by IT and in NMRI mice by intraperitoneal injection [29]. The effects of different particles (different microparticle sizes; variable crystal polymorphs α, γ, δ, and χ) were evaluated. The study concluded that the particles usually used for the manufacture of aluminum (α and γ) had no fibrotic effect, whereas other particles tested could induce this type of lesion. Moreover, this study did not show any link between cytotoxic and fibrotic effects. Potential cardiac effects of Al2O3 NPs (size 11 nm; α crystallinity; 30 mg/kg/day; over 14 days) were studied on Sprague-Dawley rats after IT [30]. This study revealed adverse effects resulting in electrocardiogram (ECG) disorders and an increase in myocardial (LDH, triglycerides, creatine phosphokinase, cholesterol, nitric oxide) and inflammatory (TNF-α) damage markers. A decrease in antioxidants was also measured (reduced glutathione and superoxide dismutase) in animal serum.

A summary of the results of in vivo studies after inhalation/instillation exposure to alumina particles is available in Table 2.


**Table 2.** Summary of animal studies.

### **4. In Vitro Studies of Cytotoxic Mechanisms Induced by Alumina Nanoparticles Exposure on Lung Cells**

A study conducted on murine fibroblasts (L929 cell line) and normal human skin fibroblasts (BJ cells) incubated with 10 to 400 μg/mL of γ-Al2O3 particles (NPs fraction of 56–91 nm and agglomerates fraction of 106–220 nm) for 24 h did not show a decrease in cell viability nor apoptosis induction. However, the authors demonstrated the same penetration dynamics of Al2O3 particles in both cell types [31]. These results were confirmed by more recent experiments on human alveolar A549 epithelial cells and skin keratinocytes HaCaT exposed for 24 h to three different Al2O3 particles (primary sizes 14 nm, 111 nm, and 750 nm; α- and α/δ crystallinities; concentrations ranging from 10 to 50 mg/L) [32]. Particles were internalized by cells in the cytoplasm but not detected in nuclei and did not exert toxicity. A comparison of the cytotoxicity induced by different metal oxide NPs (Al2O3, CeO2, TiO2, and ZnO) on human lung cell lines (A549 carcinoma cells and L-132 normal cells) concluded lower cytotoxicity of NPs of alumina compared to the other NPs tested [33]. After 72 h incubation with Al2O3 NPs, no modification of the proliferation and cell viability were observed (size 20 nm, crystallinity unknown, NPs concentrations ranging from 1 to 1000 μg/mL). Moreover, the authors did not report a significant increase in LDH nor reactive oxygen species (ROS) production. Park and colleagues studied the toxicity of three types of synthesized aluminum oxide nanoparticles (AlONPs): γ-aluminum oxide hydroxide nanoparticles (γ-AlOHNPs), γ- and α-AlONPs (diameter 180–200 nm, exposure concentrations 5 and 20 μg/mL) [34]. They exposed for 24 h six human cell lines to NPs, including bronchial epithelial BEAS-2B cells, and showed that γ-AlOHNPs induced the greatest toxicity by decreasing ATP production and normalized cell index (ICN: parameter taking into account cell number, morphology, and cell adhesion), and increasing LDH release. They postulated that low stability in biological and hydroxyl groups of γ-AlOHNPs plays an important role in their cytotoxicity and bioaccumulation. Conflicting results are available in the literature regarding the effects of alumina particle exposure on cell proliferation. A decrease in ICN of bronchial epithelial cells (cell line 169HBE14o-) was shown after exposure to Al2O3 NPs (size less than 50 nm, crystallinity unknown) for 48 h [35]. Conversely, the exposure of pleural cells (NCI-H460 cell line) at similar concentrations of alumina NPs (14 nm, unknown crystallinity) did not show any significant modification of the ICN [36].

These studies seem to show that Al2O3 particles' physico-chemical parameters (chemistry, size, etc.) and the cellular model used to determine the observed cytotoxicity. Particle size also seems to play the main role in cytotoxicity mechanisms. Indeed, human A549 alveolar epithelial cells were respectively exposed to Al2O3 NPs (sizes 10 nm and 50 nm; γ and γ/δ crystallinities) and titanium dioxide particles (TiO2, 5 nm, and 200 nm) for two and five days. Cell metabolism and cell proliferation were then studied using Alamarblue® and clonogenic assays. Contrary to Kim et al., they showed that Al2O3 NPs were more cytotoxic than TiO2 [33,37]. Smaller NPs (according to their primary size) exhibiting higher relative surface area than larger particles also induced more toxic effects, but this was not correlated with measured hydrodynamic particle sizes (diameter of the NPs and/or agglomerates in a biological medium). This suggests that the more important the specific surface area of NPs is, the more Al2O3 NPs can be cytotoxic. However, the cytotoxicity of Al2O3 NPs may not only depend on particle size. Indeed, a study investigated genotoxic effects on human fibroblasts of different particles containing alumina (NPs, microparticles, and fibers) and did not put evidence of differences in the induction of micronuclei [38]. Authors hypothesized that biological effects after exposure to particles would appear to depend on chemical composition as well as size, shape, and cell type. Recently, Bourgois et al. also exposed 24 h A549 alveolar epithelial cells to Al2O3 particles [39]. They did not show any effects of different particle sizes and crystallinities on normalized cell index, cell viability, reduced glutathione, and double DNA strand breaks.

Although results of studies on Al2O3 NPs cytotoxicity do not seem to show systematically significant decreases in cell viability, these particles can induce different biological

effects. It was shown that Al2O3 NPs (sizes 10–20 nm, crystallinity γ/<sup>α</sup>, concentrations ranging from 1 to 250 μg/mL) exposure for 24 h led to increases in mRNA and protein expression of VCAM-1, ICAM-1, and ELAM-1 in endothelial cells and increased adhesion of activated monocytes [40]. This study suggests the pro-inflammatory effects of alumina in nanoparticle form. Recently, experiments were performed on human bronchial epithelial (HBE) cells in order to characterize microRNA expression using microarrays after Al2O3 NPs exposure for 24 h (size distribution between 5 and 100 nm, unknown crystallinity, concentrations of 50 and 250 μg/mL) [41]. A homologous miRNA in Homo sapiens and Mus musculus, miR-297, was significantly up-regulated following exposures to Al2O3 NPs compared to control cells. Moreover, a few studies have reported a genotoxic potential of alumina NPs in vitro. An increased frequency of micronuclei and chromosomal aberrations has been observed on primary cultures of human fibroblasts exposed to alumina NPs (size 0.2 μm, crystallinity unknown, concentration ranging from 0.1 to 10 mg/culture flask). Nevertheless, the genotoxic effects induced were less important than those obtained in parallel with cobalt-chromium (CoCr) NPs, and no increase in DNA double-strand breaks was demonstrated in the presence of alumina [38]. Different results were obtained in another study, which showed that at low concentrations (10 μM to 1 mM), the alumina NPs (size and crystallinity unknown) were at the origin of genotoxic effects without decreasing cell viability (except at the highest concentrations) on human peripheral blood lymphocytes [42]. Indeed, these NPs increased single-strand breaks and oxidative DNA damage (2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine and 7,8-dihydro-8-oxo-2deoxyguanosine). A third study has demonstrated the induction of DNA strand breaks by Al2O3 NPs (13 and 50 nm, crystallinities unknown) after incubation with Chinese hamster lung fibroblasts [43]. Moreover, significant oxidative stress (decrease in glutathione, activity of superoxide dismutase, malondialdehyde, and total antioxidant capacity) was demonstrated after exposure to concentrations ranging from 15 to 60 μg/mL. Comparison of cytotoxic and genotoxic effects of four different NPs (oxides of cobalt, iron, silicon, and aluminum) on human lymphocytes in a recent study has, however, demonstrated that Al2O3 NPs cause less damage to DNA than the other NPs studied [44]. Nevertheless, they do significantly increase the production of reactive oxygen species and lead to a significant decrease in reduced glutathione at a concentration of 100 μg/mL. Negative results were obtained during reverse mutation tests on bacteria in the presence of alumina NPs (sizes less than 50 nm, crystallinity unknown), leading to the conclusion of an absence of mutagenic potential of these NPs [45].

Other specific effects of alumina NPs have been demonstrated in vitro. Thereby, alumina NPs (8–12 nm, crystallinity unknown, concentrations ranging from 1 μM to 10 mM) can induce a decrease in the expression of tight junction proteins [46]. A preincubation of HBMEC cells (Human Brain Microvascular Endothelial Cells, brain cell line) with glutathione blocks this effect, meaning that it could be a consequence of a phenomenon related to oxidative stress induced by exposure to NPs. Furthermore, a study carried out on erythrocytes of different species (human, rat, and rabbit) highlighted evidence of a hemolytic power of alumina NPs (13 nm, less than 50 nm, and nanofibers 2–6 nm by 200–400 nm, crystallinities unknown) [47]. Although alumina NPs are metal oxides known for their antimicrobial properties, they have only limited antimicrobial properties [48]. Only high concentrations (1000 μg/mL) have a moderate effect on bacterial proliferation (*Escherichia coli*).

A summary of the results of in vitro studies exploring the cytotoxic effects of alumina particles is available in Table 3.


**Table 3.** Summary of in vitro studies.


