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Year : 2020  |  Volume : 7  |  Issue : 2  |  Page : 84-88

Potential in the diagnosis of oxidative stress biomarkers in noninvasive samples of urine and saliva and comparison with serum of persons exposed to crystalline silica

1 Occupational Health, Student Research Committee, Hamadan University of Medical Sciences, Hamadan, Iran
2 Medical Plants and Natural Products Research Center, Hamadan University of Medical Sciences, Hamadan, Iran
3 Research Center for Biochemistry and Nutrition in Metabolic Diseases, Kashan University of Medical Sciences, Kashan, Iran
4 Modeling of Noncommunicable Diseases Research Center, School of public health, Hamadan University of Medical Sciences, Hamadan, Iran
5 Associate Professor, Research Center for Health Sciences, School of Public Health, Hamadan University of Medical Sciences, Hamadan, Iran

Date of Submission15-Jun-2019
Date of Decision19-Jan-2020
Date of Acceptance25-Feb-2020
Date of Web Publication17-Jun-2020

Correspondence Address:
Dr. Mohammad Javad Assari
Occupational Health and Safety Research Center, School of Public Health, Hamadan University of Medical Sciences, Hamadan
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/iahs.iahs_37_19

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Background: Prolonged exposure to crystalline silica (CS) (SiO2) dust enhances the production of reactive oxygen species. In many studies, oxidative stress has been measured in the serum of workers exposed to SiO2dust. Aims: We investigated the body fluids such as urine, saliva, and serum, which can provide very good results for assessing the health status of workers' exposures to SiO2dust. Materials and Methods: The oxidative stress biomarkers were evaluated in serum, urine, and saliva of 21 workers who were exposed to SiO2silica crushing factories in the Hamadan city at the west of Iran as a case group and 28 controls. Results: The level of malondialdehyde in serum, urine, and saliva was significantly higher than that in case group compared to controls (22.19 ± 8.70, 9.86 ± 5.43, and 9.41 ± 7.31 nmol/L vs. 7.30 ± 2.22, 6.79 ± 3.21, and 3.93 ± 3.73 nmol/L, respectively). In addition, the total antioxidant capacity in urine (0.23 ± 0.06 vs. 0.29 ± 0.08 mmol/L), as well as catalase in the serum and saliva of case group was lower than that compared to control group (5.46 ± 1.56 and 1.32 ± 0.55 IU/L vs. 12.55 ± 5.72 and 2.32 ± 1.53 IU/L, respectively). Conclusions: The current study indicated that chronic exposure to SiO2affects significantly on the oxidative stress biomarker levels in serum, urine, and saliva in persons exposed. Furthermore, SiO2leads to the induction of oxidative stress and decreases the activity of the antioxidant enzyme.

Keywords: Crystalline silica, oxidative stress, saliva, serum, urine

How to cite this article:
Farokhzad M, Ranjbar A, Kheiripour N, Soltanian A, Assari MJ. Potential in the diagnosis of oxidative stress biomarkers in noninvasive samples of urine and saliva and comparison with serum of persons exposed to crystalline silica. Int Arch Health Sci 2020;7:84-8

How to cite this URL:
Farokhzad M, Ranjbar A, Kheiripour N, Soltanian A, Assari MJ. Potential in the diagnosis of oxidative stress biomarkers in noninvasive samples of urine and saliva and comparison with serum of persons exposed to crystalline silica. Int Arch Health Sci [serial online] 2020 [cited 2020 Sep 22];7:84-8. Available from: http://www.iahs.kaums.ac.ir/text.asp?2020/7/2/84/286985

  Introduction Top

Silica is the most abundant mineral in the earth and a basic component of soil, sand, and rocks, including granite and quartzite. Crystalline (free silica) and amorphous silica are two types of inorganic form of silica.[1] It occurs most commonly in a crystalline form.[2] CS (SiO2) crust has various industrial applications. Respirable CS (RCS) has been classified as a carcinogen in its quartz or cristobalite forms by the National Institute for Occupational Safety and Health (NIOSH) and International Agency for Research on Cancer.[1] The health effects of exposure to silica have drawn public health concern globally.[3] Occupational exposure to RCS is a serious but preventable health hazard. Long-time exposure to RCS has long been known to cause one of the oldest known industrial diseases, silicosis, and it has been observed that there is a greater risk in workers exposed to very fine particles of CS, as found in quartz and cristobalite flours.[4] More recent studies of occupational exposure to RCS have reported some disease including chronic obstructive pulmonary disease, lung cancer,[5],[6] some autoimmune diseases,[7],[8] and nonmalignant renal disease [9],[10] in workers.

Despite the many efforts of the International Labor Organization to control the exposure of workers to SiO2, millions of workers in industries in various countries are still suffering from this issue, and silicosis, as one of the important effects of exposure to silica, is still one of the health threat in the world.[11] In some studies, the geometric mean of workers' exposure to SiO2 in stone milling and cutting, foundry, glass manufacturing, sand and gravel mining, sand blast, ceramics, bricks, cement manufacturing, and other similar factories was higher than the threshold limit value recommended by some organizations such as the American Conference of Governmental Industrial Hygienists and NIOSH.[12],[13]

Evaluating the individual exposure of workers to harmful agents such as silica is one way to proceed to control occupational diseases. When workers have an unacceptable exposure to harmful agents such as silica, their risk of disease increases. Biological monitoring (BM) is a useful method to assess the exposure of workers to chemicals in the workplace by measuring appropriate biomarkers in biological samples (urine, blood, saliva, etc.). BM can help control and reduce the exposure to chemicals and thus reduce the toxicity caused by exposure. However, until now, no valid and known biomarker has been introduced for the early diagnosis and measurement of silicosis' progress.[14],[15]

SiO2 are important occupational hazards in a wide variety of occupations, and respirable exposure to SiO2 may result in the production of reactive oxygen species (ROS).[11] A free radical is a group of atoms containing at least one unpaired electron. Unpaired electrons are highly reactive and capable of interacting with other molecules.[16] Oxidative stress due to the increased generation of ROS plays a key role in the pathogenesis and complications of many diseases.[17] Recently, oxidative stress biomarkers have been considered by the researchers in the mechanism of silicosis to find a biochemical marker. Findings suggest that SiO2 particles can be phagocytized by lung macrophages, which activate the generation of ROS.[18] Because of the key role of SiO2 in the generation of ROS, this article investigated “the body fluids such as urine, saliva, and serum in workers exposed to SiO2 dust,” while in many of the studies, oxidative stress has only been measured in the serum sample. Thus, the study can provide excellent results for assessing the health status of workers' exposures to SiO2 dust.

  Materials and Methods Top

Subject selection

In this case–control study, 21 male workers in the Hamadan city who were exposed to SiO2 in crushing factories participated as a case group. In crushing factories, silica stones are milled, powdered, and granulated, and then are supplied to the industry as Grade 1 and Grade 2 silica. The control group comprised 28 male workers who worked in the food distribution industry without exposure to CS, who were matched for age, duration of exposure, and smoking habit. We explained the purpose of the study to the workers and those unwilling to continue the study were excluded; those who were willing participated in the study with informed consent. The controls were healthy and did not consume drugs. The lifestyle and diet of the included participants were similar. The principles of the Research Ethics Committee of Hamadan University of Medical Sciences (IR.UMSHA.REC.1396.787) were followed during the entire study.

Sample collection

After assuring the satisfaction of the workers, 5 ml of fasting blood samples was taken from the participants in the study by the laboratory staff at the beginning of the shift work. Then, their serums were separated and coded. In addition, urine and salivary samples were collected from all participants and coded. The samples were kept away from direct light and stored at −20°C until analysis.[19]

Biochemical assays

Measurement of malondialdehyde level

Contents of malondialdehyde (MDA) in serum, urine, and saliva were measured spectrofluorometrically as thiobarbituric acid (TBA) reactive substances. TBA reacted with MDA and formed TBA reactant substances, as biomarkers of oxidative damage to polyunsaturated fatty acids and measured at 532 nm by a spectrophotometer.[20]

Measurement of total antioxidant capacity level

The total antioxidant capacity (TAC) was measured using the ferric-reducing ability of serum (FRAP) method. The complex among Fe2+ and tripyridyltriazine was composed, and the maximum absorption of the produced bluish complex at a wavelength of 593 nm was measured.[21]

Measurement of catalase activity

Catalase (CAT) activity in serum, urine, and saliva was determined using an assay kit according to the instructions of the company (ZellBio GmbH, Veltlinerweg 29, 89075 Ulm, Germany).

Statistical analysis

All values were expressed as means ± standard deviation (SD), and P < 0.05 was considered a statistically significant difference. Statistical analysis was done using t-test (SPSS version 16, Chicago, Ill, USA) and Graph Pad Prism version 6 (Graph Pad Software, San Diego, CA, USA).

  Results Top

Demographic parameters of the participants are presented in [Table 1]. The variables such as age (mean ± SD), smoking habit, and duration of work had no statistically significant difference between the case and control groups.
Table 1: Comparison of demographical data between case and control groups

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Concentration of malondialdehyde in the study groups

The level of MDA (nmol/L) as an oxidative stress biomarker was evaluated in serum, urine, and saliva in the case group compared with that of the control group.

As is depicted in [Figure 1], significant increases were observed in the MDA levels in the case group versus the control group in serum and saliva in the three studied matrixes (P < 0.05).
Figure 1: Malondialdehyde concentrations in serum, urine, and saliva of case and control groups. Results are represented as mean ± standard deviation

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Concentration of total antioxidant capacity in the study groups

As shown in [Figure 2], concentrations of TAC (mmol/L) in urine were statistically significantly lower in the case group when compared to that of the control group (P < 0.05). However, no statistical difference was found in the TAC level in serum and salivary samples (P > 0.05).
Figure 2: Total antioxidant capacity in serum, urine, and saliva of case and control groups. Results are represented as mean ± standard deviation

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Activity of catalase in the study groups

In serum and salivary samples, significant differences were observed in CAT (IU/L) activity regarding antioxidant biomarkers. CAT activity in urine was statistically significantly lower in case group compared to that of the control group (P < 0.05) [Figure 3]. However, no statistical difference was found in the CAT activity in the urine samples (P > 0.05).
Figure 3: Catalase activity in serum, urine, and saliva of case and control groups. Results are represented as mean ± standard deviation

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Relationship between ages, duration of exposure, and smoking habit with oxidative stress

The relationship between ages, duration of work, and smoking habit with oxidative stress factors was studied. The results showed a statistically significant negative correlation between age and plasma TAC level (r = −0.295, P = 0.04) and an inverse correlation between smoking and CAT in saliva (P = 0.007). In other cases, there was no significant relationship in the case and control groups.

  Discussion Top

SiO2 is an important material for industries, but inhaled crystalline SiO2 particles increase oxidative stress and contribute to serious diseases. In this study, we focused on the lipid peroxidation, TAC, and CAT activities in serum, urine, and saliva of the case (crushing factory workers) and control groups. Interestingly, the results of this study showed increases in oxidative biomarkers such as MDA in serum, urine, and saliva in workers exposed to SiO2 dust compared to the control group. On the other hand, exposure to SiO2 dust also reduces antioxidant biomarkers such as CAT activity and TAC levels. Concordant with these findings, previous research shown that SiO2 dust-induced oxidative toxic stress in persons exposed to crystalline silica. The study of Vallyathan et al. showed that exposure to a SiO2 particle resulted in an increased generation of ROS with enhanced levels of oxidative biomarkers such as MDA in rat lung inin vivo experiments.[22] Silica particles react with the cell membrane, leading to peroxidation of membrane lipids and damage to cell membranes.[23] In workers with cement dust-exposed silicosis, increased plasma MDA levels were reported by Orman et al.[24] Azari et al. investigated lipid peroxidation in glass sandblaster workers exposed to SiO2 aerosols through measuring serum MDA levels and indicated that serum MDA could be accepted as an indicator of oxidative toxic stress in workers exposed to SiO2 aerosols.[11] It has been shown that in the presence of water, SiO2 becomes hydrated to form silanol groups (–SiOH) that react with scavenger receptors on macrophages and activate the production of oxidative toxic stress and inflammatory cytokines.[25]

It was recently shown that inhalation of SiO2 reduced superoxide dismutase (SOD) activity as an antioxidant enzyme that these changes are consistent with the results of this research.[26] In addition, Fubini and Hubbard have shown that SiO2 can produce oxidative toxic stress by the activation of cell signaling pathways including MAPK/extracellular signal-regulated kinase (ERK) kinase, upregulation of inflammatory cytokines (e.g., tumor necrosis factor alpha and interleukin-1), ERK phosphorylation, and activation of specific transcription factors (e.g., NF-κB and AP-1).[27] Pournourmohammadi et al. demonstrated that serum levels of total thiol molecules (TTM) and TAC were significantly lower among cement plant workers in comparison to healthy controls.[18]

The present study showed a significantly inverse correlation between age and plasma TAC concentration and smoking and CAT in saliva, whereas in other cases, we did not find a correlation between case and control groups. He et al. showed that levels of serum glutathione (GSH), MDA, and SOD activity were not associated with age among both the controls and silicosis patients.[28] In addition, Kamal et al. demonstrated a nonsignificance between ages and duration of silica exposure with plasma MDA levels.[29] On investigating the effects of occupational silica exposure on oxidative stress and immune system parameters, Anlar et al. showed that there was no significant correlation between GSH levels and activities of GR, CAT, and SOD with age, smoking, and duration of exposure.[30] In the study of oxidative stress biomarkers in ceramic workers compared to a control group, the results showed a nonsignificant oxidative stress index measured with an increase in years of work.[31] Nielsen et al. revealed an association between plasma MDA and the number of hours of daily exposure to cigarette smoke, but they found no clear correlation between plasma MDA and the number of cigarettes smoked.[32]

Therefore, SiO2 can produce ROS, and thus due to the known effects of oxidative injury in the progress of silicosis, administration of antioxidant agents may be a beneficial way to prevent the progression of illness caused by silica. Furthermore, as mentioned, we investigated the body fluids such as urine, saliva, and serum in workers exposed to SiO2 dust. Although examining oxidant–antioxidant status in serum is a standard practice, but biological specimens such as urine, saliva and other non-invasive samples can be better sample than serum to diagnose and track of clinical disorders. As mentioned, saliva provides noninvasiveness and stress-free sample collection, easy and multiple sampling opportunities, decreased need for sample preprocessing, and low risk of contracting infectious organisms, and it is also an ideal biofluid for collecting samples from patients.[33] In addition, urine specimen is one of the most attractive and useful specimens for routine testing, which provides an excellent resource for the analysis of many biomarkers such as oxidative stress status, with the advantage over tissue biopsy specimens due to the ease and noninvasive nature of the collection. Therefore, saliva and urine specimens could be used in clinical practice for monitoring and diagnosis of many disease markers.

  Conclusions Top

Our results show that exposure to SiO2 dust related to increased levels of free radicals in serum, urine, and saliva. SiO2 increases the level of MDA and decreases the levels of TAC and CAT in samples from the case group. This suggests that exposure to SiO2 dust causes impairment in serum, urine, and saliva oxidant–antioxidant status. Further studies are required to comprehend the health influences and mechanisms due to human exposure to SiO2 dust.


We thank all the study participants, both controls and workers, at the Hamadan Silica Crushing factory, and the director of that factory for their kind cooperation and participation in this study. This article was supported financially by Hamadan University of Medical Sciences (no. 970128364).

Financial support and sponsorship

This article was supported financially by Hamadan University of Medical Sciences (no. 970128364).

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1]


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