Assessment of Serum Antioxidant Status in Patients with Silicosis

The Journal of International Medical Research 2010; 38: 884 889 [first published online as 38(3) 24] Assessment of Serum Antioxidant Status in Patients with Silicosis JW ZHANG 1, GC LV 1, JM YAO 1 AND XP HONG 2 1 Clinical Laboratory, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China; 2 Clinical Laboratory, Occupational Disease Hospital of the State Grid, Jiande, China Prolonged exposure to silica dust causes an imbalance in the generation of free radicals and in the antioxidant system, thereby inducing oxidative stress. The antioxidant status of 113 silicosis patients and 116 control subjects without silicosis was examined. Serum superoxide dismutase (SOD) activity and serum levels of malondialdehyde (MDA) and glutathione (GSH) were significantly higher in silicosis patients than in controls. The GSH level in patients with stage I silicosis was higher than that in patients with other stages, but there was no difference in serum MDA level and SOD activity between disease stages. The GSH level of patients who worked with air drills was significantly lower than that of patients in other occupations, whereas the MDA level was significantly elevated in patients who used air drills. Serum SOD activity did not differ significantly according to the occupational group. It is concluded that the measurement of serum SOD, GSH and MDA levels could be beneficial in the clinical evaluation of serum antioxidant status in silicosis patients. KEY WORDS: SILICA; SILICOSIS; SUPEROXIDE DISMUTASE; GLUTATHIONE; MALONDIALDEHYDE; ANTIOXIDANTS Introduction Silicosis is an occupational lung disease caused by the inhalation of crystalline silica (SiO 2 ) dust over a prolonged period. 1 It is diagnosed on the basis of a history of exposure to SiO 2 dust, relevant epidemiological data from industrial hygiene surveys, a classical presentation on dorsoventral chest radiography, clinical and laboratory examinations, and the exclusion of other similar pulmonary diseases. Inhalation of SiO 2 dust is associated with the development of silicosis, which is characterized by alveolar proteinosis and interstitial fibrosis, resulting in progressive impairment of lung function. 2,3 The potential effect of oxidative stress in silicosis has been discussed from different perspectives. 4,5 In normal physiological states, oxidation and antioxidation reactions in humans are kept in balance by a dynamic homeostatic process. A free radical theory that has been proposed to explain the mechanism of silicosis holds that lung alveolar macrophages are activated by the phagocytosis of massive amounts of SiO 2 dust, and these activated macrophages release large amounts of free radicals and various reactive oxidative species (ROS). 6 The radicals released by this process 884

overcome the radical-scavenging ability of cellular antioxidants, resulting in enhanced lipid peroxidation and damage to pulmonary structure. 7 Simultaneously, the alveolar macrophage reaction and the SiO 2 - induced immune reaction triggers the release of inflammatory factors, including nuclear factor-κb, platelet-derived growth factor and tumour necrosis factor-α, which exert additional effects on target cells. 8,9 These effects damage the lung macrophages and fibroblasts, promote collagen synthesis, and further promote the development of silicosis, resulting in a vicious circle 10 that may account for the complications of silicosis, including spontaneous pneumothorax and respiratory failure. 11 The aim of the present study was to examine the antioxidative status of silicosis patients in order to assess the degree of oxidative stress in these patients; information that should provide clues about the pathogenesis of this disease. Such information may help in the diagnosis of silicosis and in determining the best treatment strategy. Serum superoxide dismutase (SOD) activity and serum levels of glutathione (GSH) and malondialdehyde (MDA) were measured as markers of antioxidative status in patients with silicosis. Patients and methods PATIENTS This study enrolled adult males 18 years of age who had been diagnosed with silicosis, according to the Chinese National Diagnostic Criteria of Pneumoconiosis GB5906-86, and had been admitted to the Occupational Disease Hospital of the State Grid between March 2003 and May 2009. 12 Silicosis was classified into four stages (stages 0 III) according to the criteria used in China: 13 stage 0, chest radiograph not sufficient for classifying as pneumoconiosis; stage I, small rounded opacities distributed in at least two lung zones, each with a diameter 2 cm; stage II, small round or irregular opacities distributed in more than four lung zones; stage III, large opacities of length 2 cm and width 1 cm or multiple large opacities with a total area greater than the right upper lung zone. The control group consisted of healthy volunteer subjects with no history of SiO 2 dust exposure and no evidence of heart, liver, kidney or lung disease, who were recruited from the physical examination centre of The First Affiliated Hospital of Zhejiang University School of Medicine. This study was approved by the Ethics Committee of the Occupational Disease Hospital of the State Grid and was carried out in accordance with the principles of the Declaration of Helsinki. Written consent forms were obtained from all patients. SAMPLE COLLECTION Venous blood (5 7 ml) was sampled from fasting patients and serum was extracted by centrifugation. The serum samples were stored at 20 C without haemolysis for further experiments. BIOCHEMICAL ASSAYS To measure GSH, 200 µl of serum was treated with 250 µl of 0.6 mm dithiobisnitrobenzoic acid to form a yellow compound which was then measured by quantitative colorimetry at 420 nm. The nitrite formation method was used to measure SOD activity in patients serum samples; inhibition of the chromogenic reaction was monitored at a wavelength of 560 nm. 14 To measure MDA, 200 µl of serum was allowed to react with 1 ml of 6.7 g/l thiobarbituric acid and the resulting red-coloured product was quantified spectrophotometrically by measuring maximum absorption peaks at 532 nm. 885

STATISTICAL ANALYSIS Data are expressed as mean ± SD. All analyses were done using the SPSS statistical package, version 15.0 (SPSS Inc., Chicago, IL, USA) for Windows. One-way analysis of variance followed by Tukey s multiple comparison test were used to compare GSH, SOD and MDA values between the silicosis and control groups, between different stages of silicosis, and between different occupations. A P value < 0.05 was considered to be statistically significant. Results This study included 113 men who had been diagnosed with silicosis. Their mean ± SD age was 56.3 ± 12.3 years and 30 patients had stage 0, 70 had stage I, eight had stage II and five had stage III silicosis. The control group comprised 116 healthy men with no history of SiO 2 dust exposure. Their mean ± SD age was 57.6 ± 14.2 years. As shown in Fig. 1, the mean levels of serum GSH and MDA and SOD activity in the silicosis group were significantly higher than those in the control group at all stages of silicosis (P < 0.05). The GSH level in patients with stage I silicosis was significantly higher than that in patients with other stages of the disease (P < 0.01), but there was no significant difference between stages of silicosis in terms of serum MDA level or SOD activity. Comparison of serum samples of patients with different occupations revealed that the GSH level in patients who worked with air drills was significantly lower than that in patients in other occupations (P < 0.05), whereas the MDA level was significantly elevated in patients who used air drills and in those who were exposed to fuel (P < 0.05). Serum SOD activity did not differ significantly according to the occupational group (Table 1). Discussion Free radicals are generated on exposure to crystalline SiO 2 dust, either as a result of the surface activity of SiO 2 or from damage to alveolar macrophages. 15 It has been shown that ROS, such as superoxide anions, GSH concentration (mg/l) MDA concentration (nmol/ml) 20 15 10 5 0 GSH MDA SOD 120 100 80 60 40 20 0 SOD activity (U/ml) Control Stage 0 Stage I Stage II Stage III FIGURE 1: Comparison of the levels of serum markers of oxidative status (SOD, superoxide dismutase activity; GSH, glutathione concentration; MDA malondialdehyde concentration) in patients with different stages of silicosis (stages 0 III) and in control subjects without silicosis (mean ± SD; P < 0.05 compared with the control group; P < 0.01 compared with the other stages of silicosis) 886

TABLE 1: Serum markers of oxidative status in silicosis patients (n = 113) with different occupations GSH levels SOD activity MDA levels Occupation n (mg/l) (U/ml) (nmol/ml) Air driller 20 6.6 ± 1.7 97.8 ± 20.9 14.1 ± 2.7 Technician 19 10.6 ± 2.4 92.7 ± 16.9 9.4 ± 1.9 Fueller 21 11.6 ± 2.9 99.4 ± 19.1 13.6 ± 2.4 Electric welder 17 10.9 ± 2.5 96.6 ± 19.4 9.5 ± 2.3 Furnace examiner 24 10.1 ± 2.0 96.7 ± 18.8 9.2 ± 2.6 Boiler runner 12 11.1 ± 2.8 95.4 ± 18.1 9.0 ± 1.9 Data are presented as mean ± SD. One-way analysis of variance followed by Tukey s multiple comparison test were used to determine betweengroup significant differences (P < 0.05 compared with the other occupations; compared with the technician, electric welder, furnace examiner and boiler runner). GSH, glutathione; SOD, superoxide dismutase; MDA malondialdehyde. hydrogen peroxide and hydroxyl radicals, are generated through the inflammatory reaction induced by SiO 2 dust. 16 In the normal state, enzymatic antioxidants, such as SOD, and non-enzymatic antioxidants, such as GSH, serve as a defence against oxygen free radicals (OFRs). 17 Oxidative stress can be induced either by excessive production of free radicals or by the loss of defence mechanisms against antioxidants. Oxidative stress induced by SiO 2 dust is the underlying mechanism in the pathogenesis of silicosis. 18 The results of the present study indicate that OFR scavenger activity (indicated by SOD activity and GSH concentration) and the levels of lipid peroxidation products (indicated by MDA concentration) in silicosis patients were significantly higher than those in controls. We believe that the increases in SOD activity and GSH level reflect the activation of a compensatory mechanism in response to the increased level of ROS generation in silicosis, and that the elevated MDA level is clearly indicative of increased lipid peroxidation in silicosis patients. These findings are supported by those of Vallyathan et al., 4 who found increased activities of antioxidants such as SOD and GSH peroxidase in the lungs of rats exposed to crystalline silica dust. The GSH levels in red blood cells of silicosis patients have also been reported to be higher than those of control subjects. 19 Furthermore, Perrin-Nadif et al. 20 observed that SOD activity in red blood cells of underground miners was higher than that in the red blood cells of surface miners. The present study showed the serum GSH level to be significantly higher in patients with stage I silicosis compared with patients in other stages of the disease, but serum SOD activity and MDA level did not differ between the stages. Interestingly, the GSH level in red blood cells has been found to be low in the early stages and high in the late stages of pneumoconiosis in coal workers. 21 This result is contradictory to the present study, possibly because of the different kinds of dust that the subjects were exposed to. 22 Elevated levels of GSH may occur as a result of the release of cellular GSH, which scavenges free radicals, as reported by Boehme et al. 23 In the same study, it was also shown that SiO 2 dust induced a concentration-dependent increase in the GSH level in vitro and decreased cellular GSH levels in rat alveolar 887

macrophages. When comparing markers of oxidative stress in patients with different occupations, the present study found that the GSH level was significantly reduced (P < 0.05) in patients who worked with air drills and that the MDA levels were significantly higher in air drillers and fuellers compared with workers in other occupations. Thus, it could be concluded that excessive release of ROS leads to the depletion of GSH and increased lipid peroxidation in air drill workers, in whom the duration of exposure to SiO 2 dust is likely to be longer than in patients in the other occupations. In support of the present study, Zhang et al. 24 demonstrated that the GSH level decreased after SiO 2 exposure in a dose- and time-dependent manner. Thus, GSH appears to play a critical role in protecting against SiO 2 -induced cell injury. 25 In fuellers, it may be concluded that activation of the compensatory antioxidative mechanism was unable to counteract the over-production of free radicals. In conclusion, long-term exposure to SiO 2 dust may induce oxidative stress as a result of an imbalance in the generation of free radicals and in the activities of antioxidant or OFR-scavenging enzymes. The results are consistent with the premise that oxidative stress plays an important role in silicosis. Thus, it may be concluded that the measurement of serum SOD activity, and GSH and MDA levels will help clinicians to interpret dynamic changes in homeostatic oxidative and antioxidative mechanisms, and their effects on the pathogenesis of silicosis. Acknowledgements This work was supported by grants from the Natural Science Foundation (No. Y2080298) and the Traditional Chinese Medicine Foundation of Zhejiang Province, China (No. 2008CA045 and No. 2008CA054). Conflicts of interest The authors had no conflicts of interest to declare in relation to this article. Received for publication 21 December 2009 Accepted subject to revision 30 December 2009 Revised accepted 15 April 2010 Copyright 2010 Field House Publishing LLP References 1 Rimal B, Greenberg AK, Rom WN: Basic pathogenetic mechanisms in silicosis: current understanding. Curr Opin Pulm Med 2005; 11: 169 173. 2 Mossman BT, Churg A: Mechanisms in the pathogenesis of asbestosis and silicosis. Am J Respir Crit Care Med 1998; 157: 1666 1668. 3 Ding M, Chen F, Shi X, et al: Diseases caused by silica: mechanisms of injury and disease development. Int Immunopharmacol 2002; 2: 173 182. 4 Vallyathan V, Leonard S, Kuppusamy P, et al: Oxidative stress in silicosis: evidence for the enhanced clearance of free radicals from whole lungs. Mol Cell Biochem 1997; 168: 125 132. 5 Fubini B, Hubbard A: Reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation by silica in inflammation and fibrosis. Free Radic Biol Med 2003; 34: 1507 1516. 6 Gilberti RM, Joshi GN, Knecht DA: The phagocytosis of crystalline silica particles by macrophages. Am J Respir Cell Mol Biol 2008; 39: 619 627. 7 Hamilton RF Jr, Thakur SA, Holian A: Silica binding and toxicity in alveolar macrophages. Free Radic Biol Med 2008; 44: 1246 1258. 8 Langley RJ, Kalra R, Mishra NC, et al: A biphasic response to silica: I. Immuno - stimulation is restricted to the early stage of silicosis in Lewis rats. Am J Respir Cell Mol Biol 2004; 30: 823 829. 9 Polatli M, Tuna HT, Yenisey C, et al: Lung function and IFN-γ levels in the sera of silicaexposed workers. J Interferon Cytokine Res 2008; 28: 311 316. 10 Delgado L, Parra ER, Capelozzi VL: Apoptosis and extracellular matrix remodelling in human silicosis. Histopathology 2006; 49: 283 289. 11 Mohebbi I, Rad IA: Secondary spontaneous pneumothorax in rapidly progressive forms of silicosis: characterization of pulmonary 888

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