Evaluation of the toxicity of ZnO nanoparticles obtained by a chemical route on the nasal respiratory epithelium of the biomodel Mus musculus

The use and care of the animals in experimentation was carried out considering the laws established by the Colombian Government: (1) Resolution 008430 of October 4, 1993, which establishes the scientific, technical, and administrative standards for health research; Title V, Articles 87 to 93. (2) Law 84 of December 27, 1989; National Statute for the Protection of Animals. (3) Law 576 of 2000, by which the code of ethics for the professional practice of veterinary medicine and zootechnics was established” [55‐57]. The present study was endorsed by the Ethics Committee for Scientific Research of the University of Cauca as stated in the record No. 6.1 - 1.25/008 of May 9, 2018.

To define the concentrations of the synthesized ZnO to be used in the toxicity tests and to obtain representative results from them, a preliminary test was carried out. Based on information reported in the bibliography, on the average exposure limits of ZnO in the air [58, 59], a dose of 100 mg/m³ was selected, in saline solution (NaCl 0.9 % soln). This suspension was applied to six biomodels, which were exposed to it using a whole-body nebulization chamber (see Fig. 2), for 30 min and two times a day, for a period of one week. This assay allowed us to analyze the behavior of the biomodels and demonstrate histological changes in the NRE. The concentration of 100 mg/m³ caused significant changes in the behavior of the biomodels, which generated stress in them, for which three doses were established, at concentrations of 6, 15, and 40 mg/m³, to expose the Mus musculus and determine the toxic effect of the ZnO synthesized on them.

The suspensions with the defined concentrations, for the synthesized ZnO, were formed using saline solution as solvent (NaCl 0.9% soln). For this, a solution with a concentration of 100 mg/m³ was prepared and, from this, the high concentration (40 mg/m³) was obtained, from which it was started to prepare the medium concentration (15 mg/m³) and, finally, the drop (6 mg/m³).

To carry out the tests, 6-week-old male mice of the species M. musculus (CFW) were used, obtained at the Intermediate Laboratory for Preclinical Research and Murine Animal Husbandry - Animal Husbandry of the Health Faculty of the Universidad del Valle, in Cali, Colombia), which were adapted to the environment for 2 weeks, before starting the experimentation. When the biomodels reached sexual maturity (8 weeks old), they were divided into four groups (five biomodels per group) and exposed to negative control (0.9% saline solution), low dose (6 mg/m³), medium dose (15 mg/m³), and high dose (40 mg/m³), considering the concentration of the ZnO suspension to be used [58, 59].

The biomodels were exposed to the action of zinc oxide particles using a full-body inhalation chamber (made by physical engineering students from the Universidad del Cauca in Popayán, Colombia - Fig. 2), for 30 min/day, 3 days/week and in a period of 2 months. Considering the indicated exposure time and taking into account that the life expectancy of M. musculus is ~2 years, the test can be considered as chronic. Additionally, the body weight of the biomodels was recorded at baseline, mid-exposure, and before euthanasia [48, 50].

Because the nasal cavity of the biomodel is small, it was not possible to biopsy it with a cytological brush and it was necessary to euthanize the individuals. For this, a method that caused the least stress to the biomodels was selected. The intramuscular euthanasia procedure of analgesics and sedatives was chosen: ketamine (100 mg/kg) and xylazine (20 mg/kg), and their subsequent exsanguination with the purpose of eliminating or minimizing pain and stress prior to and during the procedure [60, 61].

To obtain and process the NRE of the M. musculus mouse, the protocol proposed by Young (1981) was used, with some modifications. Once the corresponding lavage of the nasal cavity was carried out, through the posterior opening of the pharyngeal canal (Fig. 3(a)), we immediately proceeded to place the head in neutral formalin plugged at 10% and allowed to set for 6–72 h. Once the fixation time had elapsed, it was decalcified [62]. This was submerged in a mixture of 15 mL of the decalcifier TBD 1- Hydrochloric acid with 5 mL of distilled water, for a period of 6 h. Subsequently, the tissues were washed twice with 70% alcohol (each for 10 min) and then returned to 20 mL of 4% formaldehyde until the sections were made for histological processing.

The decalcified head was removed from the formalin and placed on a cutting board so that the roof of the mouth (the hard palate) was facing up (Fig. 3(b)). Next, with a scalpel, two cuts were made perpendicular to the plane of the hard palate and the nasal septum: the first cut was anterior to the first palatal crest and the second cut 2–3 mm anterior to the first cut (Fig. 3(b)). A section to be processed was obtained, corresponding to the anterior part of the nasal cavity, which was labeled as Level T1.

In Fig. 4, we can see cuts made to the nasal cavity of the biomodels and that were arranged in histological plates to define the most suitable ones to carry out the corresponding analysis. The objective was to obtain representative samples of the nasal respiratory epithelium (NRE) that would allow obtaining adequate and pertinent information on the effect of the ZnO-NPs inhaled by the biomodels. For this, the information on histology of the mouse nasal cavity indicated in the book by Ruberte et al. was used as a reference [63].

Subsequently, the selected slides were observed with an optical microscope (Fig. 5), and the different types of cells that make up the nasal respiratory epithelium (Figs 5(a) to 5(d)), as well as the lamina propria, were identified. They play an important role in defending against foreign pathogens trying to enter the body [52].

The processing and staining of the NRE samples, intended for histological analysis, was carried out using the protocol established by Luna [64] and other authors such as Salazar and Moreno [65] with some modifications made by the technician from the University of Cauca, Albeiro Polanco Osorio. These procedures were developed in the morphology laboratory of the Universidad del Cauca. The modifications that were made considered the concentration of the reagents, the exposure time, and the amount used of them [64, 65]. The histological slides obtained were read by a specialist physician, member of the working group, considering the alterations experienced by the NRE samples after exposure to ZnO-NPs. The histological slides were observed with an optical microscope (Nikon eclipse 80i). Analysis was performed by evaluating squamous metaplasia, vascular congestion, and immune cells in the NRE of the biomodels [52, 66, 67].

For the statistical analysis, contingency tables were made based on the frequency of the pathologies found (squamous metaplasia, vascular congestion and immune cells) and the level of severity (not present, abundant, moderate and scarce), in other cases, presence and absence of pathology. In accordance with the nature of the data, the G test was applied to compare the treatments with the control group and it was accepted that there was an association between the variables studied when the p value was less than 0.05. The BioEstat Version 5.3 program was used [68].

The histopathological evaluation of the NRE of the M. musculus biomodels, after 8 weeks of exposure to atmospheres enriched with the synthesized ZnO-NPs, showed a temporary — reversible action of these on the nasal tissue (Fig. 9). On exposure to 0 mg/m³ (saline solution), the NRE without atypia composed of abundant hair cells lining the nasal cavity was observed (Fig. 9(a)). However, 40% were found to have polymorphonuclear (PMN) leukocytes in the lumen of the nose and only 20% had degranulating mast cells. On the other hand, exposure of biomodels to chemically synthesized ZnO-NPs induced loss of cilia or squamous metaplasia (Fig. 9(b)) and inflammatory infiltrate composed mainly of inflammatory cells, nuclear polymorphs (PMN) in intranasal clusters (Fig. 9(c)), abundant (100% of the population)—moderate and scarce (80% of the population) for the biomodels exposed to 40 mg/m³ (Fig. 9(b)), 15 mg/m³ (Fig. 9(c)) and 6 mg/m³ (Fig. 9(d)), respectively.

Another alteration that was observed in the samples analyzed was the congestion of the nasal vessels (Figs. 9(b) and (c)). Inhalation of ZnO-NPs induced moderate and scarce vascular congestion for exposures at 40 and 15 mg/m³, respectively, while for exposure at 6 mg/m³, this effect was not observed. Also, alterations in the NRE related to mast cell granulation were found to occur. It was also found that exposure to 40 mg/m³ (Fig. 9(b)) caused granulation of mast cells at different levels (abundant, moderate and scarce), while exposure to 15 mg/m³ (Fig. 9(c)) and 6 mg/m³ (Fig. 9(d)) produced abundant and moderate, mainly, and scarce effects, respectively (see Fig. 10). In addition, it was observed that when exposing the biomodels to atmospheres with 6 mg/m³ of ZnO-NPs, immune cells appeared, such as plasma cells (plasmocytes) and accumulations of lymphocytes, located under the epithelium (see Fig. 9(d)).

After the qualitative histological analysis of the different samples, to estimate the histopathological lesions caused by the inhalation of synthesized ZnO-NPs atmospheres, Fig. 10 shows the histopathological evaluation in categories of abundant, moderate, scarce or not found, of (a) squamous metaplasia, (b) nuclear polymorphs, (c) vascular congestion, and (d) mast cell degranulation. To complement the information indicated in Fig. 9, on the histopathological lesions observed in the NRE of Mus musculus that inhaled ZnO-NPs, the categorization of the alterations indicated above was carried out considering the presence or absence of: plasma cells and lymphocytes (Fig. 11).

In this study, it was evidenced that the inhalation of ZnO-NPs induced changes in the NRE of the biomodels. According to the statistical analysis, using the G test, significant differences were observed in PMN alterations, vascular congestion, and squamous metaplasia, with a p value of <0.0001 for the biomodels exposed to the inhalation of ZnO-NPs (see Fig. 10). In this same sense, the presence of immune cells, such as lymphocytes and plasma cells underlying the NRE and in the lamina propria (see Fig. 11), showed a significant difference in the exposure of ZnO-NPs with a p value of 0.0171. However, the presence of degranulating mast cells in the NRE gave, for the G test, a p value of 0.5098 for the exposure of the biomodels to ZnO-NPs. These results indicate that the alteration was not significant.

Lesions in the NRE of Mus musculus, induced by exposure to atmospheres containing the ZnO-NPs obtained in the present study, suggest that inhalation induced inflammatory reactions and morphological changes. Inflammatory reactions would be caused by the immune system, the first line of defense (innate immunity), with acute inflammation responses, in which conserved molecular patterns are recognized. This response is activated by various molecules, enzymes, and phagocytic cells, such as macrophages and neutrophils, as well as inflammatory cells (mast cells and basophils) [81].

In this stage, inflammatory cells, such as PMN leukocytes (mostly neutrophils, few eosinophils and basophils), migrate from the blood vessels and infiltrate the lamina propria and NRE, to combat potentially harmful substances to which the brain has been exposed. organism [52]. This response would be significantly induced upon exposure of the biomodels to the synthesized ZnO-NPs (Fig. 9(b)). However, it was observed that the induced alteration decreases as the exposure concentration decreases, being abundant for exposure at 40 mg/m³ and scarce at 6 mg/m³ (Fig. 10(b)).

It is necessary to remember that innate immunity is very effective. However, it does not protect the body from all infections, so adaptive immunity is activated in weeks, inducing chronic inflammation responses. In this, pathogens to which the organism has never been exposed are recognized, with the leukocytes called T or B lymphocytes coming into play, where the latter differentiate into plasma cells or plasmocytes, producing antibodies that bind to the pathogen to activate the elimination mechanism [82]. Chronic inflammatory responses were evidenced for exposure to 6 mg/m³ ZnO-NPs (Fig. 9(d)).

Another inflammatory response identified in the NRE was mast cell degranulation. Mast cells are cells resident in the connective tissue (lamina propria) of the nasal mucosa. These have several receptors on their plasma membrane that are activated when the organism comes into contact with pathogens or foreign agents, causing exocytosis of their granules found in their cytoplasm (degranulation) [81, 83]. This initiates and regulates the inflammatory response to eliminate the pathogen agent [82]. Despite the importance of mast cells in inflammatory responses, no significance was found in this study, because this alteration was also identified in the biomodels belonging to the control group (Fig. 10(d)). These results were possibly triggered by employing a whole-body spray chamber (Fig. 2), in which the biomodels caused a drastic change in their microenvironment and, consequently, in the activation of these inflammatory cells.

The morphological changes related to squamous metaplasia (Fig. 9(b)) meanwhile showed high significance in the exposure to ZnO-NPs. Furthermore, it was observed that as the exposure concentration decreased (40, 15, and 6 mg/m³), the presence of this tissue lesion was scarce (Fig. 10(a)). This histological change contemplates the replacement of the sensitive respiratory epithelium by squamous epithelium that better supports hostile environmental conditions. In this case, it would be more resistant to the injury caused by inhaled NPs. Squamous metaplasia can be identified by the disorganization of the normal respiratory epithelium, with altered polarity of the most superficial cells toward a horizontal orientation with respect to the basal lamina [84].

In this same sense, among the injuries related to exposure to chemical substances or toxic compounds, the complex network of blood vessels of the nasal cavity would be involved [85]. The blood vessels were found to be significantly congested due to the exposure to the ZnO-NPs obtained, being abundant for the exposure of the biomodels at the concentration of 40 mg/m³ and moderate for that of 15 mg/m³. Congestion of blood in the blood vessels can change the thickness of the mucosa and cause changes in airflow and nasal resistance [84].

The present research is — at least as far as that which is currently reported in the literature goes — one of the first to describe lesions in the NRE of M. musculus biomodels exposed to atmospheres containing ZnO-NPs synthesized by the Pechini method. Specifically, the histopathologies described in the results of this work, such as squamous metaplasia, vascular congestion, and inflammatory reactions (PMN, mast cells and lymphocytes) in the NRE, are frequent responses in rodents exposed to certain irritating substances or gases, and this is found documented in several studies [66]. For example, repeated exposure to formaldehyde induced alterations in the NRE of the biomodel leading to loss of the mucosal cells and the cilia (squamous metaplasia), hyperplasia or cell proliferation of the mucosal cells, focal neutrophil infiltrates, and vascular congestion [84, 86]. Other substances that, according to the literature, cause changes in hyperplastic, metaplastic and entry and acute inflammation, with the entry of neutrophils into the lamina propria or the intranasal zone, are ozone [87, 88] chlorine gas [89, 90] and ethylene [91].

In the inhalation studies carried out by other researchers, it is reported that, when the toxic substance is eliminated from the exposure medium in which the biomodel is found, the NRE does not show atypia (squamous metaplasia, hyperplasia, vascular cogestion and/or inflammatory reactions). This occurs after a recovery time of several weeks [66]. Therefore, these are considered reversible histopathologic effects from which NRE frequently suffers. Likewise, these responses of the immune system are an important mechanism, where it is verified that the organism is adequately fighting the external agent.

Although it is not easy to locate specific information on the evaluation of the toxic effect of ZnO-NPs considering the nasal cavity, there are reports of investigations that have evaluated the pulmonary toxicity by inhalation of ZnO-NPs, synthesized by chemical methods, when they pass this first defense of the respiratory system (the NRE) and that would justify the importance of the results obtained in the present work. The results of these investigations indicate that the entry of these nanoparticles, by inhalation, could generate histopathological changes in lung tissue, such as goblet cell hyperplasia, eosinophilic inflammation and fibrosis [92], cell proliferation, PMN leukocytes, foamy macrophages, and lymphocytes [47, 48]. In addition, they highlight that the interaction of ZnO-NPs with lung tissue would occur through endocytosis, where ZnO could dissolve and dissociate into Zn²⁺ [92], causing alteration in Zn homeostasis, producing reactive oxygen species (ROS), form complexes with proteins and probably alter their function [44, 93]. Additionally, these responses are considered in metal fume fever, which causes alteration in proinflammatory cytokines and recruitment of PMN leukocytes, which disappear a few days after exposure to ZnO fume [94].

In the research carried out on the effect of the inhalation of ZnO-NPs, both the exposure time and the concentration of the NPs have been considered. Adamcakova-Dodd et al. [50] showed that inhalation of ZnO-NPs, at a concentration of 3.5 mg/m³, induced a significant increase in the number of total macrophages and Zn in the lungs, but caused minimal lung inflammation, as well as little cytotoxicity or cell changes. Different results were reported by [47] where the inhalation of ZnO-NP induced foamy lymphocyte and histiocyte infiltration in the alveolar space around the terminal bronchiole, an increase in the neutrophil and eosinophil count in the bronchoalveolar fluid. They therefore concluded that inhalation of ZnO-NPs would induce inflammation in the airways. Moreover, the results obtained in this work disagree with what was reported by [49], this is because exposure to a lower concentration (6 mg/m³) induced chronic inflammatory responses in the NRE (see Fig, 9(d) and Fig. 11), which would be consistent with what was reported by Huang et al. [47], considering that NPs have the ability to travel through the bloodstream and affect other organs, including the lungs.

Other studies, such as the one carried out by Cho et al. [92], considered that ZnO-NPs have the ability to agglomerate and this should influence the toxicity of the system due to the reduction of its specific surface area and therefore its chemical reactivity. They observed that large, randomly dispersed agglomerates could trigger minor inflammatory reactions compared to well-dispersed ZnO-NPs. Therefore, they hypothesize that when ZnO-NPs are well dispersed, the induced inflammatory reactions in the tissue may be of a higher proportion [48]. This hypothesis could account for some of the results found in our research; thus, for example, that the low concentration (6 mg/m³) of ZnO-NPs was the one that induced chronic inflammatory reactions. This behavior would be more evident for this concentration because the nanoparticles would be more dispersed at the moment of inhalation and their specific surface area would be greater (increasing their reactivity), which would cause a greater adsorption in the NRE. However, a more rigorous study must be carried out to verify this hypothesis.

As indicated by the results reported in the literature on the effects of inhalation of ZnO-NPs on the lung, it is likely that exposure to ZnO-NPs involves the dissolution and dissociation processes of ZnO, such that the Zn²⁺ would promote acute inflammatory reactions with PMN recruitment and morphological changes such as squamous metaplasia and vascular congestion for high exposure concentrations (40 and 15mg/m³). Moreover, Zn²⁺ could cause chronic inflammatory reactions for the low exposure concentration (6 mg/m³), with the presence of accumulations of lymphocytes and plasma cells underlying the epithelium.

In general, the issue of air pollution and the presence of particulate matter in it has always been of great interest considering the diseases it might generate [95], specifically lung cancer [96, 97]. Interest in this topic has recently increased due to the uses that can be made of nanoparticles, for example, when their spraying on agricultural crops is required, given their potential as nanofungicides, nanopesticides, and/or nanofertilizers [4, 5, 14]. As mentioned in the introduction, this led to the structuring of a new field of knowledge called nanotoxicity [37] where different mechanisms have been evaluated and researched to account for and explain nanoparticle toxicity. Among the mechanisms discussed are direct nanoparticle association with an organism's cell surface — where the membrane can be damaged or initiate internal signaling pathways that damage the cell, cause disintegration or dissolution of the nanoparticles, leading to the release of toxic ions that impact the organism, generally through impairing important enzyme functions or through direct interaction with the cell's DNA; the formation of protein corona; and the generation of reactive oxygen species (ROS) and subsequent oxidative stress on an organism — which can also damage important enzymes or an organism's genetic material [98, 99]. Studies indicate that the factors that determine the interaction of nanoparticles with biological systems and biomolecules, specifically their toxicity, are their physicochemical properties, such as nanoparticle composition, size, surface chemistry, shape and state of agglomeration [100‐102]. Because these properties are heterogeneous in the synthesized nanoparticles, it is not easy to evaluate and understand their interaction with biological systems, adverse effects, and toxicity. In the specific case of ZnO-NPs, the nanomaterial considered in the present work, it is reported that the generation of ROS (which can be either protective or harmful during biological interactions) and therefore oxidative stress, is an important mechanism to explain their toxicity [103]. Furthermore, generation of these ROS can be mediated by the reticular defects of the nanoparticles [38, 104, 105]. The surface chemistry of ZnO-NPs is thus a critical factor in determining the cellular interactions of these nanoparticles with biological systems [106]. Considering the results obtained from the characterization of the ZnO-NPs used in the present work, in addition to the micrographs (Figs. 6(d) and 7) that show their nanometric size and state of agglomeration, the information from the Raman spectroscopy (Fig 6(b)) and XPS (Fig. 8) would indicate the presence of structural defects and for this reason, it could be reiterated that one of the most important toxicity mechanisms in the present toxicity evaluation of ZnO-NPs on the respiratory nasal epithelium would be the generation of ROS, a proposal that ought to be evaluated more systematically, in greater detail, in future research work.

In order to reach a greater understanding of the toxicity of nanoparticles, in general, and of ZnO-NPs in particular, it is therefore necessary to put forward perspectives with new approaches. We hope that the results obtained in this work are a contribution of interest to research on toxicity due to inhalation of nanoparticles and that they also motivate future work to expand our understanding of the toxicity of nanoparticles, in general.