INTRODUCTION:

Methyl methacrylate (MMA) is an acrylic resin monomer that remains widely employed in medicine, dentistry and industry. Research indicates that global demand for MMA in 2018 was 3.7 tonnes, a figure expected to increase to in excess of 4.3 tonnes in 2023 due to increasing demand for coatings and adhesives¹. Inhalation constitutes the most common means of occupational exposure to MMA². Several reports have already highlighted the negative effects of MMA vapor inhalation, such as hypersensitivity pneumonitis^3–5.

A single exposure to MMA can cause temporary pulmonary obstruction and restriction, although the symptoms will weaken and disappear on the fourth day. If exposure continues, respiratory problems may ensue⁶.

After entering the body, MMA molecules interact with cell-based nucleophilic targets resulting in the formation of reactive oxygen species (ROS)⁷. Antioxidants is a molecule capable of slowing or preventing the oxidation of other molecules^8–10, can help to protect the body from the formation of free radicals^11,12 and inhibit lipid peroxidation¹³. If the level of ROS exceeds the antioxidant defenses of the body, oxidative stress occurs inducing cell damage¹⁴ and a decrease in cell viability^15,16. Detoxification of free radicals is carried out by the body through antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) 8. SOD changes superoxide radicals into H2O2, while CAT transforms H2O2 into H2O and O2 17–19. IL-10 plays a role in controlling the nonspecific immune and cellular immune reactions mainly produced by activated macrophages²⁰. IL-10 is a cytokine with anti-inflammatory potential which enables it to maintain normal tissue homeostasis and produces a beneficial effect on airway remodeling²¹.

The negative impact of MMA monomer vapor exposure requires the implementation of precautionary measures, one of which is the provision of antioxidants. The antioxidants of natural origin have been attracted many researchers²² and a large number of plant extracts and isolates have been reported to protect against the damages caused by free radicals²³. Eucalyptol or 1,8-cineole is both an oxide and an ether, the latter of which constitutes an active ingredient containing antioxidants. This active ingredient can be found in Eucalyptus globulus and has been shown capable of inhibiting the production of ROS^24,25, decreasing NF-κB activity in vitro²⁶, inhibiting TNF-α, IL-1β and increasing anti-inflammatory cytokines IL-10²⁷.

However, the effects of eucalyptol on respiratory tract exposed to MMA remain undetermined. The purpose of this study was to determine the effect of eucalyptol on SOD and IL-10 expression in the respiratory tract of mice exposed to MMA vapor.

MATERIALS AND METHODS:

This experimental procedure was approved by the Health Research Ethical Clearance Commission of the Universitas Airlangga Faculty of Dental Medicine (Ethical Clearance Certificate Number: 041/HRECC.FODM/V/2018).

30 male BALB/c mice, aged 90 days, with a body weight of 20-30grams were selected as research subjects. After a week-long period of acclimatization, the subjects were randomly divided into five groups: two control groups (normal control group/K0 and MMA control group/K1) and three experimental groups (a 5mg/P1 eucalyptol group, a 10mg/P2 eucalyptol group and a 15mg/P3 eucalyptol group).

Initially, a glass cage previously exposed to 150ppm MMA (Merck, Schuchardt, Darmstadt, Germany) was nebulized using a nebulizer inhalator (Beurer, Ulm, Germany) at specific doses (5mg, 10mg and 15mg) of eucalyptol vapor (Merck, Darmstadt, Germany). The control group was maintained under normal conditions, while the negative control was exposed to MMA without eucalyptol.

After 120 minutes of exposure, the subjects were sacrificed by means of cervical dislocation together with two groups of control animals, their lung tissue subsequently being dissected and fixed in 10% buffered formalin. Tissue samples were processed and embedded in paraffin, sectioned at 4-5µm and prepared for immune staining with 100µg of mouse anti-human SOD monoclonal antibody (Santa Cruz, Biotechnology Inc., USA) and 100µg of mouse anti-IL-10 monoclonal (Santa Cruz, Biotechnology Inc., USA) using manufacturer-recommended dilutions.

The samples were then rinsed off with PBS both before and after being incubated with biotinylated secondary antibody (Thermo Fisher Scientific, Kalamazoo, USA). The samples were subsequently incubated with streptavidin peroxidase (Thermo Fisher Scientific, Kalamazoo, USA) for ten minutes and washed with PBS. Diaminobenzidine/DAB (Thermo Fisher Scientific, Kalamazoo, USA) was applied and incubated for 5-10 minutes in a darkened room until the desired level of staining intensity had been achieved. The samples were washed with aquabidest before being stained with Mayer Hematoxylin and incubated for 6-15 minutes at room temperature. Finally, the slides were washed with tap water.

The number of epithelial cells expressing SOD, as well as the number of macrophage cells expressing IL-10 were examined and counted using a light microscope at 400x magnification. Data was analyzed using Brown-Forsythe followed by Games-Howell test and Kruskal-Wallis followed by Mann-Whitney tests. Data analysis was performed using IBM SPSS Statistics software (IBM, Chicago, USA).

RESULTS:

The variables of this study comprised: (1) the expression of SOD and CAT in the airway epithelium and (2) the expression of IL-10 in airway macrophages (pulmonary alveoli) of the subjects were analyzed. The mean and standard deviation of SOD and IL-10 expressions can be seen in Figure 1.

Figure 1: Mean and standard deviation of SOD and IL-10 expressions. K0: normal control group; K1: MMA + Tween-Saline control group; P1: eucalyptol 5mg; P2: eucalyptol 10 mg; P3: eucalyptol 15mg

The lowest level of SOD expression was found in group P3 (15mg eucalyptol). A Games Howell test indicated that all SOD treatment groups were significantly different from the K1 group. The results of immunohistochemical preparation of SOD expression can be seen in Figure 2.

Figure 2. Immunohistochemical staining of epithelial cells of mice airways expressing SOD at 400x magnification. The brown color (indicated by the red arrow) indicates epithelial cells that expressed SOD, while the yellow arrow signifies negative expression. SOD expression was more frequently found in the K1 group, whereas the K0, P1, P2 and P3 groups contained fewer reactive epithelial cells.

The IL-10 result produced by the Kruskal Wallis test was p = 0.001 (p <0.05). The lowest number of expressions was found in the K1 group, while the highest was in the P3 group. The results of the immunohistochemical preparation of IL-10 expression can be seen in Figure 3.

Figure 3. Immunohistochemical staining of epithelial cells in mice airways expressing Il-10 at 400x magnification. The brown color (indicated by the red arrow) shows macrophage cells that expressed IL-10, while the yellow arrow indicates the negative expression. IL-10 expression in K1 group is lower than the K0, P1, P2 and P3 groups.

DISCUSSION:

The results showed that the MMA-exposed (K1) group experienced a significant increase in SOD expression (p <0.05). These results support the research findings of Soykut et al., that suggested an increase in SOD expression occurs in dental technicians exposed to acrylic/MMA through their work¹⁶. Research by Kumar et al.into 3T3 cell cultures also showed them to experience an increase in SOD post-MMA exposure²⁸. Meanwhile, a study by Aydin et al., did not identify significant differences in SOD expression between the control group and the group exposed to MMA which might be associated with significant standard deviation²⁹.

MMA can induce oxidative stress in cells through the production of ROS over a short period of time. Free radicals are unstable molecules³⁰ and the role of oxygen derived free radicals in pathogenesis of number of diseases is well known^31,32. SOD constitutes a first-line antioxidant enzyme that provides protection against oxidative damage caused by superoxide radicals. SOD catalyzes highly reactive oxygen species (O₂^-) into H₂O₂ and O₂. The ability of SOD to catalyze O₂ plays an important role in reducing ROS²⁸. In this case, SOD activity shows the ability of the body to carry out its function of scavenging free radicals. The enhanced scavenging capacity of enzymes from superoxide radicals (SOD) is an adaptation reaction which represents the main protector against damage caused by free radicals. Under normal conditions, the release of SOD will gradually increase to maintain the then-current balance between oxidation and antioxidants. The presence of higher SOD activity indicates that the scavenging ability of free radicals is also greater³³. If the level of ROS is significantly elevated, adaptability will be limited, SOD will decrease and fail to overcome these free radicals, lipid peroxidation will be formed and cells will be damaged³⁴.

Vaporized MMA can also play a role as a stressor. The research subjects of this study were terminated immediately after being exposed to MMA for two hours with the result that they might still have been in a state of acute stress. The increase of SOD identified in this study was in line with the position that acute stress causes an increase in SOD³⁵. Acute stress can activate the sympathetic adrenomodular system (SAM) axis in the hypothalamus which then activates the adrenal medulla, resulting in secretion of the hormone adrenaline that triggers a physiological reaction in the form of an increase in SOD.

The results of this study also showed that administration of 5mg, 10mg or 15mg doses of eucalyptol caused no significant difference in SOD expression from the normal control group (K0). This finding is consistent with the research results which suggested that the presence of ROS will increase the activity of SOD enzymes, while the administration of eucalyptol will weaken ROS thereby reducing SOD activity³⁶. In addition, eucalyptol also products an anti-stress effect with the result that the level of adrenaline remains constant and that of SOD becomes insignificantly different from the normal control³⁷. This finding supports the antioxidant potential of eucalyptol which can inhibit inflammation as indicated by SOD expression that does not differ significantly from that of the normal control group.

Although the main sources of IL-10 are monocytes and macrophages, there are many types of immune effector cells capable of producing IL-10 in certain contexts, including B cells, cytotoxic T cells, NK cells, mast cells and granulocytes. IL-10 is the main cytokine that plays a role in the regulation of lung inflammation during bacterial infection through neutrophil infiltration³⁸.

In this study, significant differences were found in IL-10 expression between groups (p = 0.001). MMA exposure reduced IL-10 expression. It appears that this decrease was a response to the presence of lesions originating from exposure to MMA. On the other hand, inhalation of eucalyptol can increase IL-10 expression. Following administration of a 5mg dose of eucalyptol, an increase in IL-10 occurred, although it had not yet reached normal control conditions despite being significantly different from the K1 group. At doses of 10mg and 15 mg, an increase in IL-10 expression occurred that did not differ significantly from the normal control group. This can be explained by the potential of eucalyptol to induce receptors on macrophages 2 through janus kinase (JAK). Activated JAK phosphorylates induce the inactivated signal transducer and activator of transcription/STAT-3 protein to become active. The activated STAT protein will be translocated to the nucleus via the nucleus pore, subsequently binding to the gene promoter with the result that transcription ensues. This process then produces TGF-β and IL-10, the latter of which will inhibit the processes that occur in macrophages 1³⁹.

CONCLUSION:

Eucalyptol can reduce the expression of SOD and increase IL-10 expression in airway of mice exposed to MMA vapor.

CONFLICT OF INTEREST:

The authors declare no conflicts of interest.

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Received on 27.03.2020 Modified on 14.06.2020

Research J. Pharm. and Tech. 2021; 14(6):2999-3003.

DOI: 10.52711/0974-360X.2021.00525