Ceramic Workers and the In ammatory Effects of the Silica Dust on the Nasal Mucosa Detected by Nasal Cytology: a Pilot Study


 Background. Adverse health effects due to occupational exposures are a global public health concern and have been studied for many years. Ceramic workers are occupationally exposed to a wide range of toxic substances as they manage clay (silico-aluminous feldspar mixed with sodium, potassium salts and iron oxide). The objective of this study was to assess the presence of any inflammation or alteration of the nasal mucosa of ceramic workers by nasal cytology.Materials and methods. Ceramic workers from Caltagirone (Italy) were enrolled. Nasal symptoms, atopy, health habits and workplace features were assessed by a special questionnaire, and nasal mucosa health was evaluated by nasal cytology.Results. The cytological study of the nasal mucosa showed the constant presence of abundant, thick and filamentous mucus, as well as a reorganization of the nasal cellularity with a prevalence of muciparous metaplasia and almost total absence of hair cells, for almost all of the subjects.Conclusions. A significant proportion of ceramic workers showed chronic inflammatory rhinitis on nasal cytology, with a remodelling of the nasal mucosa and thick mucus. Nasal cytology may be a helpful tool for the screening of any pathology of the upper airways.


Background
Adverse health effects due to occupational exposures are a global public health concern and have been studied for many years. A high incidence of silicosis and other pulmonary diseases in the ceramic working population has been reported from Japanese researchers in the early decades of the last century [1,2]. It is well documented that workers engaged in pottery-related industries are at great risk for pulmonary diseases, lead and other metal poisoning, as well as other toxic reactions caused by occupational exposure to clays, glazes, and furnace emissions [3]. Some reports indicate that 1,7 million workers in the United States [4] and over 3 million workers in Europe [5] are exposed to crystalline silica.
Silicosis is a well-known consequence of exposure to silica dust, and long-term exposure to crystalline silica has also been associated with the risk of developing lung cancer, pulmonary tuberculosis, and other airway diseases [6][7][8]. On the other hand, glaze or crystallinity, enamel and colours randomly used for decorations, may play a direct irritating action on airway epithelial cells. Since 1997, crystalline silica has been classi ed as a human carcinogen by the International Agency for Research on Cancer (IARC) [9], and a worldwide series of risk assessments has been conducted for respirable silica dust exposure, especially for low levels of exposure [10][11][12][13]. Although advances in occupational safety make this disorder quite avoidable, silicosis remains the most prevalent occupational disease in both developed and developing countries [14].
Health surveillance is provided to workers regularly exposed to respirable silica exclusively for lung diseases. In practice, the assessment for workers at high risk for occupational exposures, would include any respiratory questionnaire, lung function testing and a baseline chest X-ray for comparison with future chest X-rays [15][16][17]. However, there is evidence that chronic in ammatory changes in the upper airways may precede the development of lung injuries even in the absence of radiological signs or symptoms [18,19], and early screening of any changes in the normal functioning of the mucosa of the upper airways may be bene cial for the implementation of adequate preventive measures and amelioration of worker health and quality of life.
The aim of our study was to assess whether silica exposure correlated with speci c in ammatory patterns of the nasal mucosa, and nasal cytology was used as a diagnostic tool for sampling of the nasal mucosa.

Materials And Methods
Ceramic workers from the city of Caltagirone were invited to participate in the study. Caltagirone is an Italian center, that is well-renowned for its long handcrafted-ceramic tradition. Forty-ve ceramic workers volunteered, and 28 of them were selected to participate in the study according to the inclusion and exclusion criteria. The inclusion criteria were: a) workers from the hand-crafted ceramic sector; and b) workers with a minimal duration of employment of 1 year. The exclusion criteria were: a) pregnancy and lactation; b) presence of any malignancy and diseases of the immune system; c) acute and subacute infections of the airways, chronic lung diseases, chronic rhinosinusitis with or without nasal polyposis; and d) therapies affecting the health of nasal mucosa (nasal sprays with corticosteroids and/or antihistamines, oral antihistamines, NSAIDs). The study was approved by the ethical committee of AIAS di Afragola, and written consent was obtained from all the participants. The participants were administered a self-report questionnaire to investigate the presence of nasal symptoms (nasal obstruction, rinorrhea, itching and sneezing), presence of atopy and previous nasal interventions, cigarette smoking history, climate of the areas of residence and work, and workplace habits (years of exposure, type of clay and techniques of clay processing, use of plaster molding, methods of drying and ring the pieces, use of glaze, enamels or colours and techniques of decoration).
Nasal cytology: Written informed consent was obtained from any subject. Cytologic sampling was carried out by a scraping technique, i.e., by crawling a cytology plastic curette (IR Medical, Lugo, Italy), 2-3 times on the mucous surface of the central area of the inferior turbinate. The nasal mucosal cells were placed on an electrostatically charged cytology slide (Superfrost Plus Menzel-Gläser, Thermo Scienti c, Milan, Italy). The cells were then stained according to the panoptic method (3 min in pure May-Grunwald dye (Carlo Erba, Milan, Italy), 6 min in 50% May-Grunwald dye, 1 min in bidistilled water (Carlo Erba, Milan, Italy) and 30 min in Giemsa solution (Carlo Erba, Milan, Italy) diluted 1:10 v/v). The slide was then covered with a glass cover of 24 × 50 mm and observed under an optical microscope (Nikon Eclipse 50i) at 1000× oil-immersion enlargement, over fty observational elds. The images were recorded using a Nikon DS1 camera and digitized using a NIS-D elements computer support Eventually the samples were observed at microscope by the same investigator who assessed according to a 0-4 score (0= none, 1= rare, 2= some, 3= easily visible and 4= many) the presence and number of goblet cells, neutrophils, eosinophils, mast cells, bacteria and fungi [20].

The study was conducted in accordance with the International Conference on Harmonisation Good
Clinical Practice, the Declaration of Helsinki and all applicable laws and regulations.

Statistical analysis
Values are reported as a median (range) or a number (percentage), except where explicitly stated. A value of p < 0.05 was considered to be statistically signi cant. The entire analysis was performed Statistica 12 software (Statsof).

Results
Twenty-eight ceramic workers aged between 19 and 64 years old (mean age 46,28 years old) participated in the study. All of them resided in the town of Caltagirone characterized by a humid climate. The overall duration of the ceramic working period was 27,82 years. The working setting consisted of minor industries located in homes or small factories.
Of the 28 subjects, 8 (28,57%) were allergic (3 to grasses, 1 to mites, 2 to parietaria, 1 to alternaria, 1 to grass and mites), 3 (10,71%) had previous upper airway surgery (2 adenoidectomy, 1 septoplasty), 9 (32,14%) were smokers and 5 (17,85%) were former smokers. Nasal symptoms were reported by all the participants. Nasal congestion was the main symptom, followed by sneezing and itching of the nasal mucosa. In particular, the subjects who reported sneezing referred it as immediately as they entered their workplaces or during contact with the glazes and crystalline materials.
All of them worked the re ned clay by hand. 89,28% of the study population (25 subjects) reported that either the process of clay modelling, or its drying and ring, took place in the same environment, while 3 of them (10,71%) made use of an electric oven for the ring process, placed in a separate room from the workroom.
Room air extractors were present only in 5 cases (17,85%): located inside of the laboratory for 2 subjects, and for the other 3, the air extractors were placed where the electric oven was situated. Only 6 participants (21,42%) did not make use of plaster molding.
The cytological study of the nasal mucosa revealed the constant presence of abundant, thick and lamentous mucus, generally tending to be basic (Fig. 1). The mucus was detected in a lower quantity only in the samples of three subjects (10,7%), the ones with the oven in a separate room. In 10 participants (35,71%), precipitates and dust were documented (Fig. 2,3).
From a strictly cytological point of view, there was a reorganization of the nasal mucosa structure with a prevalence of muciparous metaplasia (Fig. 4) and numerous polynucleations (Fig. 3) and an almost total absence of hair cells; for rather all the subjects, 26 out of 28 or 92,85%.
Regarding the presence of in ammatory cells, 10 participants (35,71%) presented a majority of neutrophilic granulocytes (Fig. 1). Of the 8 allergic patients, nasal samples from 5 of them showed very rare neutrophils, and many lymphocytes and eosinophils were found only in a single rhinocytogram.

Discussion
The principal components of ceramic and enamel mixtures consist of crystalline silica particles (an average of 35%-38% for vitreous China and 22%-26% enamel) and other minerals such as Al, Fe, and FeCrNi alloys [21]. Occupational exposure to silica dust is well-known to be related to a higher risk of many malignant and other respiratory diseases [18,22,23].
The development of occupational diseases is highly conditioned by work environment characteristics [24] and the application of prevention measures, as predicted by the current legislation [25,26]. Studies have indicated that workers of small ceramic factories present a poorer health-related quality of life and a higher incidence of silicosis-related airway diseases, due to poor environmental conditions, irregular working hours and a high proportion of older employees [27,28]. Our data were consistent with these studies, as the majority of the participants reported that the different ceramic working processes took place in the same environment lacking protective measures: only 10,71% of our subjects made use of an electric oven placed in a separate area from the workroom, and 17,85% used an aspiration system. The only participants with a smaller quantity of mucus and in ammatory cells were those (3 out of 28) who made exclusive use of tempera colours and had a separate area provided with aspiration systems for the ring phase. Furthermore, the age of the majority of our subjects was over 60 years old.
The primary aim of this study was to assess whether silica exposure correlated with speci c in ammatory patterns of the nasal mucosa. In our sample, quite all of the subjects reported nasal congestion with itching and sneezing. It is di cult to assess whether these data are in accordance with the results of other studies. There is a lack of data about the epidemiology of nasal symptoms due to the occupational exposure of ceramic workers, as the majority of studies have focused on lower airway silica-related diseases.
The nasal mucosa is the rst target that comes into contact with airborne pollutants and the initial site of injury induced by them [29]. The mucous layer on its surface has an important function in relation to the conditioning of inhaled air and provides an adhesive surface for the deposition of inhaled particles [30].
Studies have demonstrated the potential value of nasal epithelial cells for the prediction of lung cancer risk [31]. As widely demonstrated, nasal epithelial cells, sampled either by brushing or scraping, allow the detection of toxic and in ammatory effects of airborne pollutants such as tobacco smoking, environmental carcinogens and silica [32][33][34]. To rule out the in uence of any pollen on the nasal mucosa, nasal cytology took place in November, in accordance with the data of the pollinic calendar of the area.
On nasal cytology, our ceramic workers presented a rearrangement of the respiratory epithelium of the nasal mucosa, a total absence of ciliated cells and a signi cant increase in the goblet (mucous) cell number. This pattern is described as mucous metaplasia [30]. Remodelling of the nasal mucosa has important consequences, as the diminution of ciliated cells and the increase in goblet cells cause hypersecretion of mucus and its subsequent sinonasal stagnation [29,30,32]. Reduced mucociliary transport is a major factor in the pathogenesis of rhinitis, sinusitis and other airway pathologies [35]. The presence of dust precipitates and in ammatory cells such as neutrophils and lymphocytes, is consistent with a picture of chronic in ammatory rhinitis [29]. We suggest that silica dust may act as a mucous membrane irritant, activating an in ammatory response. Since the turnover of ciliated cells is approximately 21 days, persistent in ammation may prevent the recovery of the nasal mucosa to a healthy epithelium. As a consequence of nasal obstruction, rinorrhea or sneezing occur with an important impact on worker quality of life [36].
A strong limitation of the current study is the limited sample size and the absence of a control group. A further limitation of the study was the lack of exposure data; thus, it was not possible to perform any correlation of it with the in ammatory pattern presented in the nasal mucosa.
A strength of our study is that the presence of any in ammatory pattern was analysed in the mucosa of the upper airways, rather than in peripheral blood cells or in the bronchial mucosa. In fact, the nasal mucosa is a target tissue of airborne pollutants, and its sampling can be achieved by minimally invasive methods, e.g., nasal scraping. The principal advantages of nasal scraping consist of being economical, noninvasive and easy to perform in an outpatient situation (we performed nasal scraping at the workplaces of the participants, and the smears were easier to handle than other kinds of samplings).
Other studies have con rmed the reliability of nasal cytology as a screening tool for chronic in ammatory rhinitis [32,34].
In conclusion, a signi cant proportion of ceramic workers showed a chronic in ammatory rhinitis on nasal cytology, with a remodelling of the nasal mucosa and thick mucus. Nasal cytology is a very helpful tool for the screening of early pathologic changes of the nasal mucosa to implement adequate protective measures and therapies aiming to improve worker health and quality of life. Further investigations in larger populations are required to assess the relation of the exposure levels of silica and the use of prevention equipment to the in ammatory patterns of rhinitis.

Declarations
Disclosure And Financial Support: All the authors do not have con icts of interest. This study did not receive any nancial support.  Figure 1 Numerous neutrophils (arrow) in heavily blue coloured (cross) smear because of the mucus. Observation by optical microscope. Magni cation at 1000 X oil immersion. Col. MGG.