The Effect of titanium dioxide nanoparticles on the activity of salivary peroxidase in periodontitis patients

: Background:The technology of nanoparticles has been expanded to many aspects of modern life. Titanium dioxide nanoparticles were of many nanomaterials utilized in biomedical applications. The interactions between nanoparticles and proteins are believed to be the base for the biological effect of the nanoparticles. The oxidation reaction of many sub-stances is catalyzed by oxidizing enzymes called peroxidases. The activity of salivary peroxidase is elevated with periodontal diseases. the aim ofthis study is to examine the action of titanium dioxide nanoparticles on salivary peroxidase activity.Material and method75 participants were enrolled in this study — Periodontitis group with 44 participants and the non-per-iodontitis group with 31 participants. The participants' age range was 35 to 50 years for both groups. The clinical parameters of plaque index, gingival index, probing pocket depth and clinical attachment level were used in this study to determine the presence or absence of the periodontal disease. Unstimulated saliva was collected from all participants and analyzed for the activity of peroxidase enzyme under the effect of titanium dioxide nanoparticles. Re-sultsThe Periodontitis group showed higher peroxidase enzyme activity than the non-perio-dontitis group and the activity of salivary peroxidase showed no correlation with the clinical parameters. Titanium dioxide nanoparticles increased salivary peroxidase activity. Conclu-sionThis study demonstrated that the solid surface of nanoparticles could induce changes in the attached protein molecule which in turn causes changes in the effect of the nanoparticles on living tissue or organism. The titanium dioxide nanoparticles Play a role in increasing the activity of salivary peroxidase within the saliva of chronic periodontitis patients.


Introduction
Periodontitis is a wide-spreading disease characterized by the pain-free and sluggish spread of the condition. This disease starts with a plaque under the gingiva and is altered by the immune response. The disease can occur in different age groups but is most common in adults. It causes loss of the periodontium, which in time leads to tooth mobility and loss (1,2) . bodily fluids such as saliva and tears. In addition, it can be found in cells, removing free radicals with the assistance of H2O2. The two structures of peroxidases, which are salivary peroxidase and myeloperoxidase, are detected in the whole saliva and play an important role in the defense mechanism (5) . Nanomaterials consist of components less than 100 nm in at least one dimension 6 . Titanium dioxide nanoparticles (TiO2 NPs) have many distinctive qualities and properties such as compatibility with living tissue and optical properties 7 and many studies showed its antibacterial activity (8,9) when added to dental materials, Therefore TiO2 NPs have been investigated profusely in recent years to make advances in dentistry. te aim of this study was to analyze the action of TiO2 NPs on peroxidase enzyme activity in patients suffering from chronic periodontitis.

Sample selection:
In this study, two study groups were recruited. The first group (group 1) consisted of 44 participants (periodontitis group), while the second group (group 2) consisted of 31 participants (non-periodontitis group). The total number of participants was 75 and the age range was from 35 to 50 years. The collection of the samples started in October and finished in December of 2017. The participants were patients seeking treatment in the Department of Periodontics at the College of Dentistry, University of Baghdad. Both consent forms and ethical approval were acquired for this study.

Clinical examination:
The periodontal parameters (gingival index (10) , plaque index (11) , bleeding on probing, clinical attachment level, and probing pocket depth were used for the assessment and diagnosis of the periodontal condition of the participants.

Collection of Saliva
The spitting method for the collection of whole unstimulated saliva was used. Patients were asked to rinse their mouth first, then wait a few minutes before spitting into a plane tube to collect 5 ml of saliva. This was done at least one hour after the participant's last meal. The time of sample collection was between 9 a.m.-11 a.m. After the collection of the sample; it was centrifuged for 15 minutes at 2500 rpm. A layer called supernatant is formed and then collected and stored in an Eppendorf tube at -20˚C.

Laboratory procedures:
a. Saliva sample volume determination: To determine the best saliva volume to obtain the optimum activity of peroxidase, different volumes of saliva samples were tested (20,40, 60, 80, and 100µl).
In this experiment, different volumes of saliva were collected (20, 40, 60, 80 and 100 µl) to determine the best saliva volume to measure the activity of salivary peroxidase. The optimum enzyme activity for this experiment was found to be (100 µl).
b. Characterization of Titanium dioxide nanoparticles: TiO2 Nanopowder was obtained from Hongwu international group Ltd, Guangdong, China. An ultraviolet-visible spectrophotometer (PG Instruments Limited/ United Kingdom) was used to determine the absorption spectra for the TiO2 NPs solution used in this study. The measurement was done at room temperature. The size and structure of the Tio2 NPs in the samples were determined using a transmission electron microscope TEM (Philips CM10).
c. Salivary Peroxidase Assay: The colorimetric method was used to determine the activity of the peroxidase enzyme in saliva. In this study, 4-aminoantipyrine was used as a hydrogen donor. The H2O2 decomposes through the incubation period causing an elevation in absorption at λ= 510 nm, which helps in determining the activity of the

d. Preparation of TiO2 NPs solution:
The first step was to prepare a stock solution of TiO2 NPs (300 µg/ml). A solvent of 3:1 water to ethanol was used to dilute the stock solution to different concentrations (20, 40, 60, 80, 100, and 120 µg\ml). The best concentration was found to be 120 µg\ml.

Statistical analysis:
Statistical package for social sciences (SPSS) and Microsoft Office Excel were used to analyze the collected data. The significant difference was assesedusing the student t-test (P < 0.05). The Pearson correlation coefficient was also used.

Clinical findings 13
The mean values and standard deviations (SD) of Peroxidase activity for group 1 were highly significant compared to group 2 (p< 0.001).
The mean values and SD of Peroxidase activity for both groups are shown in table1: The correlation was non-significant when comparing the activity of peroxidase and both plaque index (PLI) and gingival index (GI) in group 1. A weak negative and non-significant correlation was determined for GI and a non-significant correlation for PLI in group 2. Results are as illustrated in the table below:  TEM was used to identify the structure and Nano size measurement of TiO2 NPs in the samples ( figure   2). The average diameter of the particle size was found to be < 30nm.

The effect of TiO2 NPs on Peroxidase enzyme in saliva
The differences in the activities of peroxidase enzyme (mean ± SD) for the studied groups are shown in Table (3) below. The results conveyed that the activity of peroxidase in saliva samples from group 1 with TiO2 NPs was greater than the enzyme activity in samples without TiO2 NPs with a statistically high significance difference (p-value< 0.001), as shown in the table below. Similarly, a highly significant difference was also found After comparing the enzyme activity in group 2 with and without TiO2 NPs.    TiO2 NPs= titanium dioxide nanoparticles

Discussion
Nowadays, TiO2 NPs were incorporated in many products used in daily life which made it necessary to evaluate the action of this type of nanoparticle on living systems (12) .
The chemical evaluation revealed that TiO2 NPs caused an elevation in peroxidase activity. Al-Rubaee et al. in 2016 showed that Total salivary peroxidase was activated significantly (p˂0.001) by TiO2 NPs 13 . The surface of the nanoparticles can cause changes in the form of the protein when it attaches itself to the nanoparticle surface. This change in the protein form can lead to a change in the protein's function. This changes the bio reactivity of the nanoparticles (14) .
Many studies showed evidence of conformational changes when enzyme interacts with NPs, such as a study on Zinc oxide NPs in which it was found that the NPs modify the secondary structure of lysozyme.
The enzyme keeps its catalytic activity and resists denaturation in the presence of these NPs (15) .
In another study to determine the effect of zinc oxide nanoparticles on peroxidase enzyme activity, an inhibition action on peroxidase enzyme activity was noticed for this type of nanoparticles (16) . The activity of peroxidase was found to be elevated in periodontitis compared to non-periodontitis patients, with a significant difference. Similar results were obtained in another study by Al-Rassam et al. in 2017 (17) in which a comparison was made between salivary peroxidase activity in the chronic periodontitis group and a control group, the enzyme activity was elevated in the chronic periodontitis group compared to the control group and with a significant difference. Another study found that salivary peroxidase activity significantly increases with inflammation and reduces after oral hygiene measures (18) .
In another study, Glutathione peroxidase in saliva and gingival tissues of subjects with and without chronic periodontitis was evaluated and it was found that there was an increase in Glutathione peroxidase level in saliva and inflamed gingival tissue (19) .
The elevated peroxidase level in the saliva and tissue of the gingiva of patients suffering from periodontitis can be accredited to the scavenging of the redundant lipid peroxidation products at the inflammatory sites (20) .
Also, the elevated level of reactive oxygen species (ROS) formed may have caused oxidative stress, which lead to an increased need for peroxidase generation to establish the ROS-AO balance to prevent tissue damage (19) .
In this study, a non-significant correlation between the gingival index and the activity of salivary peroxidase in group1 and group2, a similarly non-significant correlation was found between plaque index and the action of salivary peroxidase in both groups (P> 0.05). This is, in contrast, to a study by Dagar et al. in 2015 (21) in which a significant correlation was found between the activity of peroxidase and PLI. This difference in results could be accredited to the variation in saliva-collecting methods, the number of participants in each study, and analysis methods.
In this study, TiO2 NPs caused an increase in the action of the peroxides enzyme. Peroxidase enzyme level in the saliva of patients suffering from periodontitis was significantly higher than patients without periodontitis disease.
However, more research are needed to overcome the limitations of this study. Some of these limitations, such as the limited study sample size, the time constraints, and the technique-sensitive method for preparing the nanoparticle solution, can be addressed in future studies for more precise results.

Conclusion
The solid surface of nanoparticles can induce changes in the attached protein molecule which in turn causes changes in the effect of the nanoparticles on living tissue. The titanium dioxide nanoparticles play a role in increasing the activity of salivary peroxidase within the saliva of periodontitis patients.