The environment has a significant impact on human health. In terms of global disease burden, air pollution accounts for more than one-third of deaths from lung cancer, stroke, and chronic respiratory disease, and nearly one-quarter of deaths from ischemic heart disease. The combined effects of indoor and outdoor air pollution have resulted in about 7 million deaths from air pollution annually (Ferrero et al., 2017; WHO, 2015). Pregnant women are one of the vulnerable and susceptible groups, against health-threatening factors such as toxic compounds present in polluted air. Considering the health of pregnant women who are exposed to toxic pollutants in the air, it is important because in addition to the health of the mother, the health of her fetus is also under threat (Mannucci and Franchini, 2017). According to the results of some studies, pregnant women in industrial areas due to exposure to toxic pollutants were prone to preterm delivery and consequently increased risk of fetal injury, low birth weight and intrauterine growth restriction (Phatrabuddha et al., 2013; Ritz and Wilhelm, 2008; van den Hooven et al., 2011).

The petrochemical industries, as one of the sources of air pollution, play an important role in the production and release of toxic pollutants, including BTEX to the environment, and have adverse effects on public health, especially pregnant women living in the vicinity of these industries. Benzene, toluene, ethylbenzene and xylene (BTEX) are a group of  Volatile Organic Compounds (VOCs) (Tsangari et al., 2017),  that due to their adverse effect on human health are classified as hazardous air pollutants (Rafiee et al., 2018). International Agency for Research on Cancer (IARC), classified benzene as a human carcinogen (group 1) and ethylbenzene as a possible carcinogen (group 2) ((IARC), 2000). The source of BTEX emissions may be outdoor activities including traffic congestion and industrial processes (Bailey and Eggleston, 1993; Bauri et al., 2015; Truc and Kim Oanh, 2007), and also indoor sources such as smoking, chemicals used in the structure of furniture and interior decoration, color, and glue (Hazrati et al., 2015; Singh et al., 1992). Therfore, people living in polluted areas and close to industrial areas are more likely to be exposed to BTEX and consequently these population face a greater risk from exposure to these chemicals (Crebelli et al., 2001; Sutic et al., 2016).

The effects of exposure to BTEX compounds on human health can also be categorized as short-term effects including nausea, headache and dizziness, eyes and skin irritation, throat and nose irritation, and asthma exacerbation (Blount et al., 2006; Mohammadyan et al., 2016; Rafiee et al., 2019; Zhou et al., 2011), or in the form of long-term effects including leukemia and congenital birth defect, impact on central neural system (CNS), as well as adverse effects on the respiratory system (Rafiee et al., 2019; Sekhar and Subramaniyam, 2014). Evaluation of biological evidence showed that benzene had an adverse effect on intrauterine growth during pregnancy or in another study indicated the possible role of benzene in adverse effects at birth (Chen et al., 2000; Khan., 2007). In addition to the above-mentioned effects, overall, toxic air pollutants induce oxidative stress in the lung, causing chronic respiratory tract and ultimately systemic inflammation, which increase the levels of immune factors (cytokines) in the circulatory system and can, in severe cases, cause inflammation of the brain (Block and Calderón-Garcidueñas, 2009; Guxens et al., 2012). Cytokines are soluble hormone-like proteins that affect the activity, differentiation, proliferation and survival of immune cells and regulate the activity of other cytokines by increasing (pro-inflammatory cytokines) or decreasing (anti-inflammatory cytokines) (de Oliveira et al., 2011; Dinarello, 2007; Tayal and Kalra, 2008).

Some cytokines, including, interleukin-6 (IL-6) and Tumor necrosis factor-a (TNF-α), play key roles in acute inflammatory interactions. Interleukin-6, an anti-inflammatory cytokine with  multiple functions is produced by macrophages, monocytes, and T cells. These cytokines play an important role in regulating immune reaction and inflammatory responses (El-Khier et al., 2013; Ma et al., 2017). Tumor necrosis factor-a (TNF-α) is produced by the monocytes and is one of the most important proinflammatory cytokines that contribute to increased bronchial excitability or airway remodeling in asthmatic patients (Akdis et al., 2011), promotes tumor growth and migration (Esquivel-Velázquez et al., 2015), and or in the development of liver inflammation (Tuncer et al., 2003). In general, proinflammatory cytokines such as TNF-α, after binding to target cells, induce protein tyrosine phosphorylation and ultimately inhibit the activity, proliferation, or differentiation of different cells during immune responses (de Oliveira et al., 2011; Dinarello, 2007). Nowadays, the use of Human Biological Monitoring (HBM), as a viable and reliable approach to assess human exposure to chemicals and measure their metabolites, using biomarkers in human specimens, and also, as an important tool for environmental protection and policy-making of human health is considered (Sciences, 2005; WHO, 2015). The use of urine samples and serum samples, respectively, for the measurement of BTEX compounds and immune factors has been used in the current study.  Since pregnancy is a vital period for the mother and fetus has been associated with a number of physiological changes in pregnant women, therefore, these changes can affect the toxicity of chemicals into the body. Considering the importance of maternal and fetal health during pregnancy as an indicator of health development assessment in a community, this study aimed to exposure assessment of pregnant women living in the vicinity of petrochemicals to BTEX compounds and evaluation of its possible impact on immune factors, IL-6 and TNF-α were assessed. The results of this study can be used as a useful information tool for health organizations to adopt management policies and strategies.

2. Materials and method

2.1. Study design and participants

This study was designed to exposure assessment of pregnant women living in the vicinity of petrochemicals to BTEX compounds and evaluation of its possible impact on immune factors, IL-6 and TNF-α. The present study was conducted from August to October 2019. All participants in this study lived in their current place of residence for more than 10 years. The study population consisted of 200 pregnant women who were divided into case (n = 110) and control (n = 90) groups. The demographic data and medical records of the participants in this study are shown in Table 1. According to Figure 1, the distance between the case group and the control group from the petrochemical company was 2 and 30 km, respectively. For a comprehensive assessment of changes in serum concentration of immune factors, subjects were randomly selected from each trimester including first trimester (n = 10), second trimester (n = 15) and third trimester (n=5). Blood and urine samples were obtained from pregnant women for a period of 2 weeks. The implementation protocol for this cross-sectional study, Urine and Blood samples collected from pregnant women referred to Chavar comprehensive health center (Iran, Ilam) was approved by the Ethics Committee of the Shiraz University of Medical Science (Number), and written informed consent was obtained from all pregnant women included in the present study.

2.2. Clinical and labratoary data

2.2.1. Biomonitoring protocol of urinary BTEX

Sample collection, storage and preparation

Urine samples were obtained from pregnant women who referred to Chavar comprehensive health center (Iran, Ilam). Urine samples were collected in a special encoded container, and stored at 4°C in the freezer until sent to the laboratory. Once every two days, the collected samples were sent to the laboratory in a cold box containing dry ice to analyze BTEX compounds. All glassware used in this study was sterilized prior to the extraction process of BTEX compounds. To prevent contamination of glass containers, they were first sterilized by ultrasonic process and then immersed in 20% nitric acid solution for 24 hours. Finally, they were washed with distilled water and incubated at 180°C for 5 hours.

Sample preparation and headspace solid-phase micro extract

In order to prepare the urine samples for quantitative analysis, 2 ml of each urine sample was transferred to 4 ml glass vials containing 1 mg sodium chloride. To reduce the urine foaming, a small amount of 0.3 – 0.4 µl of antifoam (Antifoam C, Restek, USA) was injected into each vial. Using a micro-syringe, 1 µl of methanolic solutions of benzene-d6, toluene-d8, m-xylene-d10, and naphthalene-d10 was added to each vial as internal standard. Immediately to seal the vials, a magnetic crimp cap with a a silicone-PTFE septa product from Sigma Aldrich was used. To create a uniform solution, samples were shaken and then stored at -20°C. Headspace Solid-PhaseMicro-Extraction (HS-SPME) was performed for analysis of non-metabolite BTEX compounds in urine samples. Prior to any extraction operation, the vials were incubated at a controlled temperature of 37°C for 20 min at a stirring rate of 750 rpm. The analytics were extracted using a PDMS 75 μm fiber for 5 min and then by insertion of the fiber into the chromatographic injection port, thermal desorption for 3 min was done.

In order to separate the analytics, by Gas Chromatograph (GC, Agilent 7890N, Agilent Co.), Helium gas at a purity of 99.999% and mass flow rate of 1 ml/min through a DB-5MS analytical capillary column (30 m length, 0.25 mm diameter and 0.25 μm film thickness) for separation of the analytics by The GC device was used. Finally, mass spectrometry (MS, Agilent 5975C, Agilent Co.) was used to detect and quantify the constituents of BTEX.

All of the BTEX reference materials and solutions from Sigma Aldrich (Austria) were purchased, in the range of purities from 99% to 99.8%. In parallel to each urine sample, a blank sample containing distilled water was prepared for each participant using the same materials and solutions according to the same protocol described for urine. Blank samples were analyzed similar to urine samples and if BTEX is present, the BTEX concentration is subtracted from the values ​​in the urine samples. In the present study, the detection limit (LOD), quantitative limit (LOQ) and matrix effects were used to control the method used. The LOD and LOQ related to the BTEX compounds are shown in Table 2. The limit of detection (LOD) of the assay for each BTEX compound was calculated according to the following formula: LOD = (3Sy-a)/b, where “Sy” is the standard error associated with each BTEX compound, “a” and “b” are intercept and slope, respectively.

2.2.2. Determination of IL-6 and TNF-α in serum

The blood samples were withdrawn from pregnant women within 2 weeks under complete aseptic conditions had been collected in a Vacationer (Gel & Clot Activator, AB MEDICAL Co., Ltd, South Korea). Approximate 3 mL blood samples were obtained from each participant. After each sampling, the samples were immediately sent to the laboratory for serum preparation and separation. Then, for serum preparation, the samples were centrifuged at room temperature at 3500 rpm for 15 min and immediately stored at -80°C for measuring Interleukin-6 and TNF-α. The Serum concentrations of Interleukin-6 and TNF-α were carried out by using the Enzyme-Linked Immunosorbent Assay (ELISA) according to the manufacturer’s instructions, kit supplied by Biosource International (CA, USA). The concentrations of measured cytokines were expressed in units of pg/mL. In order to, IL-6 assay, a human IL-6 high-sensitivity ELISA kit (sensitivity: <0.16 pg/mL) with the minimum detectable level < 1 pg/mL and subsequently for TNF-α assay, a human TNF-α high-sensitivity ELISA kit (sensitivity: <0.5 pg/mL) with the minimum detectable level 0.09 pg/mL were used. Finally, the absorbance of the samples was measured at a wavelength of 450 nm by ELISA reader. Due to increased levels of IL-6, under inflammatory and infectious conditions (Buttaro et al., 2010; Honda et al., 1990) and In order to investigate the relationship between Immune factors and the effects of exposure to BTEX compounds, women with chronic inflammatory diseases such as rheumatoid arthritis (12 patients), Granulomatous mastitis (2 patients), Type 2 Diabetes Mellitus (6 patient), Periodic fever (1 patient), Reproductive system infections (16 patient), Inflammatory bowel diseases, (13 patient) and women taking antibiotics within one week (30 people) were excluded from the study.

Statistical analysis

Statistical Package for the Social Science (SPSS) software, version 22.0 software (IBM Co., USA) was used for analysis. Descriptive analysis of the results were performed using frequency, percentage, mean and standard deviation. Prior to any statistical tests, the normality of the data was evaluated first by Skewness and Kurtosis and then by Kolmogorov-Smirnov test. All data obtained in this study did not have a normal distribution (P < 0.05). Due to the lack of data normality, the Kruskal-Wallis test was used to compare the difference between the concentrations of BTEX compounds measured in urine samples of pregnant women. Then, Spearman correlation coefficient analysis was used to investigate the relationship between the concentration of cytokines measured in blood samples and the concentration of different BTEX compounds in urine samples. The optimal cut-off value of the cytokines was estimated using Receiver Operating Characteristics (ROC) analysis. Area under the curve (AUC), calculated as sensitivity and specificity. The Area Under the Curve (AUC) was calculated to determine sensitivity and specificity.


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