Serum Prolidase Activity in Silicosis and Its Relationship with Oxida-Tive Stress and Inflammation Markers

1. Introduction

Silicosis is an interstitial lung disease resulting from fibrosis of the lungs caused by inhalation of crystalline silicon dioxide or silica. It is still one of the most important occupational diseases worldwide. Even when silica exposure is eliminated, the disease can progress to progressive massive fibrosis and lead to impaired lung function [1]. In silicosis, direct cytotoxicity, oxidant production, proinflammatory cytokine and reactive oxygen species (ROS) release and growth factor release in the pathological process stimulate scar formation and lung fibrosis [2].

The enzyme prolidase catalyzes the hydrolysis of dipeptides containing proline or hydroxyproline in the carboxyl-terminal position. Proline is recycled and used in new protein synthesis, while hydroxyproline is excreted in the urine. Since proline and hydroxyproline constitute approximately 25% of the amino acids in collagen structure, prolidase plays an important role in collagen degradation [3].

Prolidase is involved in collagen metabolism, new matrix formation, and cell growth. Prolidase, therefore, plays an important role in various physiological processes such as wound healing, inflammation, angiogenesis, cell proliferation, and carcinogenesis [3,4]. Deviations in prolidase activity may be a useful marker in cancers and many conditions characterized by fibrosis [4]. In people with liver disease, serum prolidase activity is increased, especially in the early stage of fibrosis, and it has been suggested that plasma prolidase activity may be useful in assessing fibrotic processes in chronic liver disease in humans [5]. The relationship between lung fibrosis and prolidase in silicosis patients is unclear and has never been studied. It is thought that prolidase enzyme may be a useful marker in silicosis as in other diseases with fibrosis.

Prolidase activity in bronchial asthma [6] and chronic obstructive pulmonary disease COPD [7,8] is lower in patients with dilated cardiomyopathy, pancreatitis and pancreatic cancer. Prolidase activity has been reported to be different in each chronic disease; prolidase activity is decreased in patients with chronic uremic type 2 diabetes mellitus [9] and is increased in chronic liver diseases [10].

Although there are studies evaluating oxidative stress and inflammatory markers in silicosis, there is no study evaluating serum prolidase activity. In this study, we aimed to determine whether serum prolidase activity is a new marker in the diagnosis and follow-up of silicosis. We also aimed to contribute to understanding pathophysiologic mechanisms in silicosis by determining oxidative stress parameters, inflammatory markers and serum prolidase activity. With the help of the identified markers, we aimed to contribute to the prevention of the disease by terminating the exposure at an early stage and taking preventive measures.

2. Methods

This study was approved by the Clinical Research Ethics Committee of Diskapi Yildirim Beyazit Training and Research Hospital (Date and Number: March 27, 2017, 36-13). All participants signed informed consent.

2.1. Study Population and Study Procedures

The study population consisted of silica-exposed workers referred to the Ankara Hospital for Occupational and Environmental Diseases, one of the three reference centers in Turkey, by an occupational physician or pulmonologist with suspicion of silicosis. Subjects who did not have a chronic disease had a history of exposure, did not have silicosis, and worked in the same workplace were included in the exposure group. Age- and gender-matched healthy subjects without dust exposure were selected as the control group. Our study consisted of 113 silicosis patients, 70 workers with silica exposure who did not develop the disease and 53 healthy volunteers, totaling 236 male participants.

Those with chronic diseases characterized by fibrosis in the lung or any organ (interstitial lung disease, scleroderma, liver cirrhosis, ankylosing spondylitis, rheumatoid arthritis, cardiomyopathy), those taking medications that affect prolidase levels, active infections and malignancies were excluded. All participants completed a detailed clinical questionnaire including age, gender, height, weight, smoking habit, chronic disease, pulmonary symptoms, use of personal protective equipment, total exposure time, and occupational history.

Pulmonary function tests were performed with appropriate techniques in all participants. Chest radiographs of all participants were evaluated by two pulmonologists with International Labor Office (ILO) radiography reading training certificates. Any disagreements were resolved by consensus. We defined silicosis as associated radiological signs (ILO classification of ≥1/0) and radiological findings on high-resolution computed tomography (HRCT) in a worker exposed to silica [11].

2.2. Biological Sampling

Venous blood was collected from each volunteer as a biological sample. Blood was collected in silicone-coated tubes and protected from light. Blood samples were centrifuged at 2000 rpm for 15 minutes. The serum samples obtained were stored at -80°C until the analysis day. Serum samples stored at room temperature on the day of the experiment were used to study biomarkers.

2.3. Determination of Serum Prolidase Activity and Other Parameters

All kits (Prolidase, catalase (CAT), 8-hydroxy-2-deoxyguanosine (8-OH-dG), glutathione peroxidase (GPx), fibroblast growth factor (FGF), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin (IL) 1α, 1β) were obtained from the Cloud-Clone Corp. (Wuhan, China); other additional reagents were purchased from Cayman Chemical (Michigan, USA). Biotek Instruments (Vermont, USA) was employed to make spectrophotometric measurements.

The levels of prolidase, inflammatory and oxidative stress parameters were measured using commercial kits using the ELISA technique. The absorbance of each well was measured at 450 nm.

2.3. Statistical Analysis

All statistical analyses were performed in R Statistical Software version 4.1.2. (The R Foundation for Statistical Computing, Vienna, Austria; https://www.r-project.org). Before the analyses, the normality of the data was checked by Shapiro-Wilk’s normality test and Q-Q graphs, and Levene’s homogeneity test checked the homogeneity of group variances. The findings of the numerical variables in the study were presented as mean ± standard deviation or median and quartiles (first quartile - third quartile), and categorical variables were presented as frequency (n) and percentage. The presence or absence of statistically significant differences in the demographic, clinical, functional and radiological findings of the silicosis, exposure and healthy control groups were evaluated by one-way ANOVA followed by Tukey HSD multiple comparisons; Welch’s F test followed by Games-Howell multiple comparisons; Kruskal Wallis H test in groups that did not show normal distribution, followed by multiple comparisons with Dunn’s test with Bonferroni correction for parameters found significant. Independent sample t-test or Mann-Whitney U test was used to evaluate whether there was a statistically significant difference in the prolidase, oxidative stress and inflammatory findings of silicosis, exposure and healthy control groups and simple and complicated silicosis groups. The groups ‘ working times and mask use rates were compared with the Pearson chi-square test. In addition, Spearman’s rho correlation coefficient was used to determine whether there was a statistically significant relationship between serum prolidase activity and demographic, clinical, functional, and radiologic findings.

3. Results

3.1. Demographic and Clinical Characteristics

Table 1 presents the characteristics of the study population. Although silicosis patients were older than the silica-exposed group, there was no statistical difference in age between both groups and healthy controls (p >0.005). Although smoking rates in the control group were similar to those in the general population, smoking rates were significantly higher in the silicosis and silica-exposed groups (p < 0.001). There was a statistical difference between the groups in the duration of study exposure (p = 0.004). Patients with silicosis had longer working hours than the silica-exposed group. While 43.4% of patients with silicosis had worked for 15 years or more, this rate was 27.1% in the group with exposure. In the silicosis group, 39.8% always wore masks, while this rate was 51.4% in the group with exposure.

According to the lung function test findings of the subjects, forced expired volume (FEV1) and forced vital capacity (FVC) values were significantly lower in both the silicosis and exposure groups compared to healthy controls (p<0.001). Peak expiratory flow (PEF) and FEV1/FVC values were not statistically different between the groups. The diffusion capacity of carbon monoxide (DLCO) was found to be lower in the silicosis group compared to exposed individuals, but no statistical significance was found (p >0.005).

According to the ILO classification, 61 (54%) of 113 silicosis patients were classified as Category 1, 31 (27.4%) as Category 2, and 21 (18.6%) as Category 3. The predominant opacity type was p opacity, with 57.5%, and Category 1 was the most common type of profusion. Large opacities were detected in 19 (17%) silicosis patients. Thirteen large opacities were A, 3 were B, and 3 were C. The mean ILO profusion score of silicosis was 6 (5-9).

3.2. Prolidase, Oxidative Stress, and Inflammatory Parameters

Although serum prolidase activity was higher in the silicosis and silica-exposed group than in healthy controls, the difference was not statistically significant. Serum prolidase levels were highest in silicosis, while prolidase levels tended to increase after silica exposure (Table 2).

Serum superoxide dismutase (SOD) and GPx levels were significantly lower in the exposure and silicosis groups compared to the control group. Although CAT was lower than the control, it was not statistically significant. Similarly, 8-OH-dG, an indicator of silica-induced DNA damage, and inflammatory parameters TNF-α, FGF, IFN-γ, and IL-1α were found to be significantly higher in the exposure and silicosis groups compared to the control group (Table 2).

A comparison of prolidase, oxidative stress and inflammatory parameters in simple silicosis and complicated silicosis groups is given in Table 3. Prolidase levels were higher in subjects with complicated silicosis than those with simple silicosis. However, the difference between the groups was not statistically significant. Except for GPx, there was no statistically significant difference between the two groups in all other parameters.

A positive significant correlation was found between serum prolidase activity and IFN-γ only (Spearman’s rho=0.140, p=.032). On the other hand, no statistically significant correlation was found between serum prolidase activity and other biochemical parameters (all p>0.05).