Just Accepted by International Journal of Radiation Biology
A cross-sectional study on oxidative stress in workers exposed to extremely low frequency electromagnetic fields Li Li, De-fu Xiong, Jia-wen Liu, Zi-xin Li, Guang-cheng Zeng, Hua-liang Li Doi: 10.3109/09553002.2015.1012304
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Abstract Purpose: To investigate whether extremely low frequency electromagnetic field (ELF-EMF) exposure could induce oxidative stress in workers performing tour-inspection near transformers and distribution power lines. Materials and Methods: Occupational short-term “spot” measurements were performed. In total, 310 inspection workers exposed to ELF-EMF were selected as the exposure group and 300 logistical staff as the control group. Plasma total antioxidant capacity (T-AOC) and glutathione peroxidase (GPx) activity were tested by the colorimetric method. Superoxide dismutase (SOD) activity was tested using the xanthine oxidase method. Plasma malodialdehyde (MDA) concentration was determined with a thiobarbituric acid assay. The micronucleus cell frequency (MCF) and Micronuclei frequency (MN) were also tested for genotoxic assessment. Results: No significant changes of enzyme activities or MDA concentration were found. Neither the frequency of micronucleus lymphocytes nor micronuclei frequency changes were statistically significant. Conclusion: Continual ELF-EMF exposure might not induce oxidative stress in workers from a power supply bureau.
© 2015 Informa UK, Ltd. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted articles have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication.
A cross-sectional study on oxidative stress in workers exposed to extremely low frequency electromagnetic fields
Li Li1, De-fu Xiong2, Jia-wen Liu1, Zi-xin Li2, Guang-cheng Zeng1, Hua-liang Li1
1
Electric Power Research Institute of Guangdong Power Grid Corporation, Guangzhou,
China, 2Guangdong
Huianhengda Management Consulting Co.,Ltd,
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Guangzhou, China Corresponding Author: Li Li, Electric Power Research Institute of Guangdong Power Grid Corporation, No.8 Shuijungang Dongfengdong Road, Guangzhou, Guangdong, 510080, China. Tel:+86-020-34063102. Fax:+86-020-89021031. E-mail: [email protected] Sort Title: Oxidative stress in workers exposed to ELF-EMF Abstract Purpose: To investigate whether extremely low frequency electromagnetic field (ELF-EMF) exposure could induce oxidative stress in workers performing tour-inspection near transformers and distribution power lines. Materials and Methods: Occupational short-term "spot" measurements were performed. In total, 310 inspection workers exposed to ELF-EMF were selected as the exposure group and 300 logistical staff as the control group. Plasma total antioxidant capacity (T-AOC) and glutathione peroxidase (GPx) activity were tested by the colorimetric method. Superoxide dismutase (SOD) activity was tested using the xanthine oxidase method. Plasma malodialdehyde (MDA) concentration was determined with a thiobarbituric acid assay. The micronucleus cell frequency (MCF) and Micronuclei frequency (MN) were also tested for genotoxic assessment. Results: No significant changes of enzyme activities or MDA concentration were found. Neither the frequency of micronucleus lymphocytes nor micronuclei frequency changes were statistically significant. Conclusion: Continual ELF-EMF exposure might not induce oxidative stress in workers from a power supply bureau.
Keywords: ELF-EMF, occupational exposure, oxidative stress, genotoxicity
Introduction ELF-EMF, which are produced by alternating current at 30~300 Hz, are regularly generated from power distribution networks and electric appliances from which the frequency generated is 60 Hz or 50 Hz. Electric and magnetic fields of 50 Hz measured beneath energized power transmission lines are generally in the ranges of 1-10 kV/m and 1-10 μT, respectively. However, they can reach 11 kV/m and 100 μT, or even much higher in the environment of
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transformer stations, electric enclosures or magnetotherapy. [Lacy-Hulbert et al., 1998; Petrucci et al., 1999; Goraca et al., 2010; Cam et al., 2011]. The possible relationship between ELF-EMF exposure and the interferences on biological processes has been a durable subject for a long time, but the effects of ELF-EMF on health are still ambiguous [World Health Organization (WHO), 2007]. In 2002, the International Agency for Research on Cancer (IARC) recognized extremely low frequency magnetic field (ELF-MF) exposure as a possible human carcinogen [IARC, 2002]. Recently, the most significant focus of ELF-EMF exposure has been placed on its carcinogenicity. There are considerable concerns about whether ELF-EMF exposure could result in or promote the occurrence of cancer arising from increasing exposed populations, such as brain cancer [Baldi et al., 2011], breast cancer [Forssén et al., 2000] and childhood leukemia [Kheifets et al., 2010]. Therefore, related researches on whether ELF-EMF exposure could induce oxidative stress have increased correspondingly, since oxidative stress could give rise to DNA damage which might further initiate the process of cell malignant transformation [Zwirska-Korczala et al., 2005; Martínez et al., 2012]. This might be one of the underlying mechanisms of the contribution of
ELF-EMF exposure to carcinogenesis [Wolf et al., 2005]. Previous studies reported that ELF-MF exposure could modulate the expression or activity of redox-related proteins, resulting in reactive oxygen species (ROS) production in cells [Türközer et al., 2008; Frahm et al., 2010]. ROS generation processes initiated by oxidative stress play a decisive role in important biological processes, such as inflammation, cell division, differentiation, apoptosis, and cellular senescence [Jarrett et al., 2012]. ROS is a known causative factor in
Int J Radiat Biol Downloaded from informahealthcare.com by Nyu Medical Center on 02/12/15 For personal use only.
the oxidative damage of cellular structures and macromolecules such as lipids, proteins, and nucleic acids [Birben et al., 2012]. Organisms harbor large quantities of antioxidants to prevent or repair the damage caused by ROS, and to regulate redox-sensitive signaling pathways [Di Loreto et al., 2009]. Intracellular ROS concentrations are under the control of antioxidant enzymes, and the intrinsic antioxidant enzyme system has been identified as a defense mechanism that protects cells against oxidative injury [Akdag et al., 2010]. Three primary antioxidant enzymes, which are necessary for life in all oxygen-metabolizing cells, might play predominant roles in the antioxidant processes initiated by ELF-MF exposure including SOD, catalase (CAT) and GPx [Sharifian et al., 2009; Akdag et al., 2010; Ciejka et al., 2011]. The WHO Research Agenda (2007) recommended in vitro studies of ROS evaluation as a high-priority line of inquiry regarding the research field of biological effect of EMF [WHO, 2007]. However, studies found that antioxidant enzyme activities and protein expression involved in cellular redox regulation did not appear to be affected by ELF-MF [Benfante et al., 2008; Hong et al., 2012]. Absence of genotoxicity in cells exposed to ELF-MF seems to support the view that ELF-MF exposure may not induce excessive
oxidative stress [Stronati et al., 2004]. Moreover, some of the previous epidemiological studies on this subject likely suffered from shortcomings like small sample number and no specific known EMF exposure [Kheifets et al., 2010], thus, a clear answer to whether EMF exposure could induce oxidative stress in the human body remains to be elucidated. More evidence from other types of cells, in vivo tests and especially exposed population are required to clarify the controversial results mentioned above.
Int J Radiat Biol Downloaded from informahealthcare.com by Nyu Medical Center on 02/12/15 For personal use only.
The current study is intended to investigate whether ELF-MF exposure could induce oxidative stress in workers regularly performing tour-inspection close to voltage transformers and distribution power lines. The current study aims to provide some clues and evidences for clarifying the ambiguous correlation between ELF-EMF exposure and the oxidative stress. Materials and methods Subjects A total of 700 subjects from a power supply bureau were recruited and designated into the exposure (n=360) and control (n=340) group depending on whether there was occupational history of ELF-EMF exposure. The research protocol was approved by the institutional review board (The Ethics Committees of Electric Power Research Institute of Guangdong Power Grid Corporation, Guangzhou, Guangdong, China). Informed consent and Health Insurance Portability and Accountability consent were obtained from each subject. All the subjects were required to complete a questionnaire in at least 15 mins, which mainly included questions about nationality, smoking, alcohol, green tea drinking, medicine intake and medical inspections like chest perspective, X-Ray based imagery and barium meal. In consideration of the
effects of confounding factors， the subjects with histories of present illness, past illness and family heredopathia of mental and cardiovascular diseases were not eligible. At last, a total of 610 eligible workers were included in the study in which 300 workers from administrative staff and logistical personnel without ELF-EMF exposure were placed in the control group and 310 workers that regularly performed tour-inspections on foot close to voltage transformers and distribution power lines were placed in the exposure group.
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ELF-MF Measurement and assessment Given that the effects of ELF-EMF were mainly attributed to ELF-MF, and ELF-MF exposure have been recognized as a possible human carcinogen [International Commission For Non Ionizing Radiation Protection (ICNIRP), 2001; IARC, 2002], the dose of extremely low frequency electric fields (ELF-EF) for each exposed subject was ignored in the current study. The “short-term” measurement procedure was established and performed similarly to a previous study [Cam et al., 2011]. All the short-term spot measurements of ELF-EMF were made using a PMM 8053A portable field meter with an EHP50 three-axial probe (PMM Construction Center for Electronic Radio Measurements, Cisano Sul Neva, Savona, Liguria Italy). The measurement spots in the 500 kV and 220 kV transformer stations started 2 m from the authorized entrance of the areas and dotted at the outdoor routes of tour-inspections at 5 m apart and 1.5 m high from the ground. Other indoor spots dotted in rooms where the inspection staff took rest while they were off inspection were also measured at 1.2 m high from the ground. The measurement lasted for approximately 2 min at each spot, and the reference root mean square (RMS) level of ELF-MF was recorded. Environmental
non-occupational ELF-MF exposure levels of both control and exposed groups from the living areas and urban offices were not measured. In total 200 spots of ELF-MF levels in the environment where the control subjects (logistical staff) worked were measured. The measuring method was the same as that applied in the measurement spots in 500 kV and 220 kV areas. All exposed subjects were asked to record their main activities during work and non-working periods. The ELF-MF exposure dose was calculated and
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expressed as 8 h time weighted average magnetic flux intensity (TWA): TWA =
[ B1 × t1 + B2 × t2 + B3 × t3 ] (μT). Where B and B represented the average 1 2 8
magnetic flux intensities of 500 kV and 220 kV areas. B3 represented the magnetic flux intensity in the rooms (within the station) where inspection staff took rest when they were off tour-inspection. t1, t2 and t3 represented the corresponding time spent in 500 kV, 220 kV areas of inspection and offices. Blood Samples A 5-cm3 venous blood sample was collected from each subject by a practiced licensed phlebotomist using a 21-gauge needle. Light tourniquet pressure was applied in all subjects to assist venipuncture. Blood was collected into tubes containing anticoagulant ethylene diamine tetraaceticacid potassium (EDTA-K+) and then centrifuged for 10 min at 5,000×g to remove platelets. The plasma was then removed, aliquoted and stored at -80°C until all the samples were collected and then the oxidative parameters were tested. All blood samples were collected at approximately the same time of day when tour-inspection staff completed their inspection work, and also in case the circadian variations might exert effects on oxidative stress levels. Determination of Plasma T-AOC level
T-AOC was determined with an Ultra-spect spectrophotometer (Pharmacia, Houston, Texas, USA) following the procedure described by Benzie and Strain [Benzie et al., 1996] with modifications. A colored ferrous-TPTZ (tripyridyl-triazine) complex was generated at low pH while Fe3+ ion was reduced to Fe2+ ion, which resulted in an increase in absorbance at 593 nm. Serum proteins and low molecular weight -SH rich compounds did cause much interference, because they had very low activity in this method. Briefly,
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50 µl plasma were diluted with 50 µl deionized water and then added to 900 µl of ferric reducing antioxidant power (FRAP) reagent Sample absorbance at 593 nm was continuously measured for 10 min at 37°C. In total, 100 µl of water was added to the control (blank) samples. A calibration curve was performed for aqueous solutions of ferrous sulfate (FeSO4) (50-1200 µM). Results were expressed in μM. Determination of GPx Activity GPx activity was measured by following the GPx Assay Kit instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, the reaction mixture contained 2.7 mL of potassium phosphate buffer (0.067 M, pH 6.6), 0.1 mL EDTA (15 mM), 0.1 mL of Nicotinamide Adenine Dinucleotide Phosphate (NADPH) (6 mM in 1% [w/v] sodium bicarbonate), and 50 µL of sample. A change in color was monitored by recording absorbance (340 nm) at 1-minute intervals for 3 mins. One unit of the enzyme activity (U/min/mg) was expressed as n moles of nicotinamide adenine dinucleotide phosphate (NADPH) oxidized/min/mg protein using the molar extinction coefficient of NADPH (6.22 ×106 M−1cm−1). Measurement of Serum SOD Activity
SOD activity in the serum of all subjects was measured by the xanthine oxidase method using a SOD Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer’s instructions. The xanthine oxidase system generates a superoxide anion radical which oxidates hydroxylamine and simultaneously generates prunosus nitrite compounds in the presence of a color-developing agent. The SOD activity of each sample is in a linear correlation with absorbance, and expressed as U/ml.
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Cytokinesis-block Micronucleus Test A micronucleus test was performed referring to the standard procedure [Fenech et al., 2007], with minor modifications, after cytokinesis was blocked. Briefly, 4.7 ml of serum-free cell freezing medium 1640 (Roswell Park Memorial Institute (RPMI)-1640) containing 10% fetal bovine serum (FBS), 1% L-glutamine and antibiotics (100 IU/ml penicillin, and 100 μg/ml streptomycin) was added to 0.3 ml whole blood for lymphocytes culture. Next, 2% phytohemagglutinin (PHA) was added to stimulate cultures. Two independent cultures for each subject were incubated at 37°C, 5% CO2 and 90% humidified atmosphere for 44 hrs. Next, 6 μg/ml cytochalasin-B was added to the cultures to block cytokinesis for another 28 h (whole culture time 72 h). The cells were harvested and subjected to a hypotonic state. Cells were then prefixed with ice-cold fixative solution (5:1 v/v methanol: acetic acid), and then washed and fixed with 10 ml of fresh fixative for 1 h on ice. Cell suspensions were finally dropped onto slides and then stained for 5 min with 4% Giemsa in Sörensen’s phosphate buffer (0.067 M Na2HPO4/KH2PO4, pH 6.8) after dried. Microscopy was performed at 400× magnification and then confirmed at 1,000× magnification. All slides were analyzed blindly. The
MCF in 500 cells of each independent culture with well-preserved cytoplasm was scored and calculated as: micronucleus cell number/all the cells counted×100%, MN calculated as: micronucleus number/ all the cells counted×100%, according to established criteria. Statistical Analysis The group average of each serum oxidative stress marker and the ratio of micronucleus were calculated and presented as mean±standard deviation (SD),
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and range for 95% confidence interval was included. The total group averages and averages in different exposure durations and exposure level groups of serum oxidative stress markers were stratified, analyzed, and compared using a one-way analysis of variance (ANOVA) to determine if any statistically significant difference existed. The differences of MCF and MN between the control and exposure group were compared with a u test in Poisson Distribution. All P values less than 0.05 were considered statistically significant. All the statistical analyses were performed using SPSS 11.0 software (SPSS.Inc, Chicago, Illinois, USA).
Results The ELF-MF intensity in the route of tour-inspections nearby 500 kV and 220 kV transformers and distribution lines which were selected randomly from the 11 of the total 22 transformer stations was measured respectively. In the 500 kV area, the intensity of magnetic filed intensity at the spots ranged from 0.62 to 30.19 μT and the median ELF-MF level was 18.72 μT. The magnetic filed intensity in the 220 kV area ranged from 0.51-60.11 μT, and the median ELF-MF level was 17.72 μT. In the environment of the control, Max and Min ELF-MF levels were 0.72 and 0.07 μT respectively, and the median ELF-MF
level was 0.21 μT was much lower than that in both 220 kV and 500 kV. Individual ELF-MF exposure doses were calculated and expressed as 8 hr TWA according to the exposure time provided by the subjects’ detailed record of work days. The average TWA was 7.3 μT ( 1.56 ~ 26.33 μT ) and the subjects were subgrouped by cumulative ELF-MF exposure dose: low (< 1.56 μT ), middle ( 1.56 ~ 7.3 μT ) and high ( > 7.3 μT). Total oxidative stress level analysis
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Overall, 560 male workers were included in oxidative stress level analysis including 280 exposed individuals and 280 control individuals. The 280 male workers in the exposure group (average age 30.9±5.6) were divided into 2 sub-groups by exposure duration, 154 male workers in ＜5 y exposure group and 126 male workers in ≥5 y exposure group. There were 50 female workers (average age 29.8±4.5) in this study, including 20 females in the control group and 30 females in the exposure group. The total differences of T-AOC, SOD, MDA, GPx, MCF and MN levels between the control and exposure groups were analyzed with a u-test. The average levels of T-AOC, SOD, MDA, GPx, MCF and MN in the control vs exposure groups were respectively:15.3 μM vs 15.1 μM, 108.4 U/ml vs 107.4 U/ml, 4.5μM vs 4.3 μM, 200 nmol NADPH oxidized/min/mg protein (U/min/mg) vs 197 U/min/mg, 2.6% vs 2.8%, 2.7% vs 3.0% . No statistical difference was found (P > 0.05). Considering gender as an influence， the male workers and female workers were analyzed separately. The results showed the levels of T-AOC, SOD, MDA and Gpx between female exposure and control groups were not statistically different. The difference among the sub-groups of female workers was not analysed due to the limited number. When all the male
subjects were sub-grouped, the changes of oxidative stress biomarkers level or activity among different total service time (Control,
A cross-sectional study on oxidative stress in workers exposed to extremely low frequency electromagnetic fields Li Li, De-fu Xiong, Jia-wen Liu, Zi-xin Li, Guang-cheng Zeng, Hua-liang Li Doi: 10.3109/09553002.2015.1012304
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Abstract Purpose: To investigate whether extremely low frequency electromagnetic field (ELF-EMF) exposure could induce oxidative stress in workers performing tour-inspection near transformers and distribution power lines. Materials and Methods: Occupational short-term “spot” measurements were performed. In total, 310 inspection workers exposed to ELF-EMF were selected as the exposure group and 300 logistical staff as the control group. Plasma total antioxidant capacity (T-AOC) and glutathione peroxidase (GPx) activity were tested by the colorimetric method. Superoxide dismutase (SOD) activity was tested using the xanthine oxidase method. Plasma malodialdehyde (MDA) concentration was determined with a thiobarbituric acid assay. The micronucleus cell frequency (MCF) and Micronuclei frequency (MN) were also tested for genotoxic assessment. Results: No significant changes of enzyme activities or MDA concentration were found. Neither the frequency of micronucleus lymphocytes nor micronuclei frequency changes were statistically significant. Conclusion: Continual ELF-EMF exposure might not induce oxidative stress in workers from a power supply bureau.
© 2015 Informa UK, Ltd. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted articles have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication.
A cross-sectional study on oxidative stress in workers exposed to extremely low frequency electromagnetic fields
Li Li1, De-fu Xiong2, Jia-wen Liu1, Zi-xin Li2, Guang-cheng Zeng1, Hua-liang Li1
1
Electric Power Research Institute of Guangdong Power Grid Corporation, Guangzhou,
China, 2Guangdong
Huianhengda Management Consulting Co.,Ltd,
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Guangzhou, China Corresponding Author: Li Li, Electric Power Research Institute of Guangdong Power Grid Corporation, No.8 Shuijungang Dongfengdong Road, Guangzhou, Guangdong, 510080, China. Tel:+86-020-34063102. Fax:+86-020-89021031. E-mail: [email protected] Sort Title: Oxidative stress in workers exposed to ELF-EMF Abstract Purpose: To investigate whether extremely low frequency electromagnetic field (ELF-EMF) exposure could induce oxidative stress in workers performing tour-inspection near transformers and distribution power lines. Materials and Methods: Occupational short-term "spot" measurements were performed. In total, 310 inspection workers exposed to ELF-EMF were selected as the exposure group and 300 logistical staff as the control group. Plasma total antioxidant capacity (T-AOC) and glutathione peroxidase (GPx) activity were tested by the colorimetric method. Superoxide dismutase (SOD) activity was tested using the xanthine oxidase method. Plasma malodialdehyde (MDA) concentration was determined with a thiobarbituric acid assay. The micronucleus cell frequency (MCF) and Micronuclei frequency (MN) were also tested for genotoxic assessment. Results: No significant changes of enzyme activities or MDA concentration were found. Neither the frequency of micronucleus lymphocytes nor micronuclei frequency changes were statistically significant. Conclusion: Continual ELF-EMF exposure might not induce oxidative stress in workers from a power supply bureau.
Keywords: ELF-EMF, occupational exposure, oxidative stress, genotoxicity
Introduction ELF-EMF, which are produced by alternating current at 30~300 Hz, are regularly generated from power distribution networks and electric appliances from which the frequency generated is 60 Hz or 50 Hz. Electric and magnetic fields of 50 Hz measured beneath energized power transmission lines are generally in the ranges of 1-10 kV/m and 1-10 μT, respectively. However, they can reach 11 kV/m and 100 μT, or even much higher in the environment of
Int J Radiat Biol Downloaded from informahealthcare.com by Nyu Medical Center on 02/12/15 For personal use only.
transformer stations, electric enclosures or magnetotherapy. [Lacy-Hulbert et al., 1998; Petrucci et al., 1999; Goraca et al., 2010; Cam et al., 2011]. The possible relationship between ELF-EMF exposure and the interferences on biological processes has been a durable subject for a long time, but the effects of ELF-EMF on health are still ambiguous [World Health Organization (WHO), 2007]. In 2002, the International Agency for Research on Cancer (IARC) recognized extremely low frequency magnetic field (ELF-MF) exposure as a possible human carcinogen [IARC, 2002]. Recently, the most significant focus of ELF-EMF exposure has been placed on its carcinogenicity. There are considerable concerns about whether ELF-EMF exposure could result in or promote the occurrence of cancer arising from increasing exposed populations, such as brain cancer [Baldi et al., 2011], breast cancer [Forssén et al., 2000] and childhood leukemia [Kheifets et al., 2010]. Therefore, related researches on whether ELF-EMF exposure could induce oxidative stress have increased correspondingly, since oxidative stress could give rise to DNA damage which might further initiate the process of cell malignant transformation [Zwirska-Korczala et al., 2005; Martínez et al., 2012]. This might be one of the underlying mechanisms of the contribution of
ELF-EMF exposure to carcinogenesis [Wolf et al., 2005]. Previous studies reported that ELF-MF exposure could modulate the expression or activity of redox-related proteins, resulting in reactive oxygen species (ROS) production in cells [Türközer et al., 2008; Frahm et al., 2010]. ROS generation processes initiated by oxidative stress play a decisive role in important biological processes, such as inflammation, cell division, differentiation, apoptosis, and cellular senescence [Jarrett et al., 2012]. ROS is a known causative factor in
Int J Radiat Biol Downloaded from informahealthcare.com by Nyu Medical Center on 02/12/15 For personal use only.
the oxidative damage of cellular structures and macromolecules such as lipids, proteins, and nucleic acids [Birben et al., 2012]. Organisms harbor large quantities of antioxidants to prevent or repair the damage caused by ROS, and to regulate redox-sensitive signaling pathways [Di Loreto et al., 2009]. Intracellular ROS concentrations are under the control of antioxidant enzymes, and the intrinsic antioxidant enzyme system has been identified as a defense mechanism that protects cells against oxidative injury [Akdag et al., 2010]. Three primary antioxidant enzymes, which are necessary for life in all oxygen-metabolizing cells, might play predominant roles in the antioxidant processes initiated by ELF-MF exposure including SOD, catalase (CAT) and GPx [Sharifian et al., 2009; Akdag et al., 2010; Ciejka et al., 2011]. The WHO Research Agenda (2007) recommended in vitro studies of ROS evaluation as a high-priority line of inquiry regarding the research field of biological effect of EMF [WHO, 2007]. However, studies found that antioxidant enzyme activities and protein expression involved in cellular redox regulation did not appear to be affected by ELF-MF [Benfante et al., 2008; Hong et al., 2012]. Absence of genotoxicity in cells exposed to ELF-MF seems to support the view that ELF-MF exposure may not induce excessive
oxidative stress [Stronati et al., 2004]. Moreover, some of the previous epidemiological studies on this subject likely suffered from shortcomings like small sample number and no specific known EMF exposure [Kheifets et al., 2010], thus, a clear answer to whether EMF exposure could induce oxidative stress in the human body remains to be elucidated. More evidence from other types of cells, in vivo tests and especially exposed population are required to clarify the controversial results mentioned above.
Int J Radiat Biol Downloaded from informahealthcare.com by Nyu Medical Center on 02/12/15 For personal use only.
The current study is intended to investigate whether ELF-MF exposure could induce oxidative stress in workers regularly performing tour-inspection close to voltage transformers and distribution power lines. The current study aims to provide some clues and evidences for clarifying the ambiguous correlation between ELF-EMF exposure and the oxidative stress. Materials and methods Subjects A total of 700 subjects from a power supply bureau were recruited and designated into the exposure (n=360) and control (n=340) group depending on whether there was occupational history of ELF-EMF exposure. The research protocol was approved by the institutional review board (The Ethics Committees of Electric Power Research Institute of Guangdong Power Grid Corporation, Guangzhou, Guangdong, China). Informed consent and Health Insurance Portability and Accountability consent were obtained from each subject. All the subjects were required to complete a questionnaire in at least 15 mins, which mainly included questions about nationality, smoking, alcohol, green tea drinking, medicine intake and medical inspections like chest perspective, X-Ray based imagery and barium meal. In consideration of the
effects of confounding factors， the subjects with histories of present illness, past illness and family heredopathia of mental and cardiovascular diseases were not eligible. At last, a total of 610 eligible workers were included in the study in which 300 workers from administrative staff and logistical personnel without ELF-EMF exposure were placed in the control group and 310 workers that regularly performed tour-inspections on foot close to voltage transformers and distribution power lines were placed in the exposure group.
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ELF-MF Measurement and assessment Given that the effects of ELF-EMF were mainly attributed to ELF-MF, and ELF-MF exposure have been recognized as a possible human carcinogen [International Commission For Non Ionizing Radiation Protection (ICNIRP), 2001; IARC, 2002], the dose of extremely low frequency electric fields (ELF-EF) for each exposed subject was ignored in the current study. The “short-term” measurement procedure was established and performed similarly to a previous study [Cam et al., 2011]. All the short-term spot measurements of ELF-EMF were made using a PMM 8053A portable field meter with an EHP50 three-axial probe (PMM Construction Center for Electronic Radio Measurements, Cisano Sul Neva, Savona, Liguria Italy). The measurement spots in the 500 kV and 220 kV transformer stations started 2 m from the authorized entrance of the areas and dotted at the outdoor routes of tour-inspections at 5 m apart and 1.5 m high from the ground. Other indoor spots dotted in rooms where the inspection staff took rest while they were off inspection were also measured at 1.2 m high from the ground. The measurement lasted for approximately 2 min at each spot, and the reference root mean square (RMS) level of ELF-MF was recorded. Environmental
non-occupational ELF-MF exposure levels of both control and exposed groups from the living areas and urban offices were not measured. In total 200 spots of ELF-MF levels in the environment where the control subjects (logistical staff) worked were measured. The measuring method was the same as that applied in the measurement spots in 500 kV and 220 kV areas. All exposed subjects were asked to record their main activities during work and non-working periods. The ELF-MF exposure dose was calculated and
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expressed as 8 h time weighted average magnetic flux intensity (TWA): TWA =
[ B1 × t1 + B2 × t2 + B3 × t3 ] (μT). Where B and B represented the average 1 2 8
magnetic flux intensities of 500 kV and 220 kV areas. B3 represented the magnetic flux intensity in the rooms (within the station) where inspection staff took rest when they were off tour-inspection. t1, t2 and t3 represented the corresponding time spent in 500 kV, 220 kV areas of inspection and offices. Blood Samples A 5-cm3 venous blood sample was collected from each subject by a practiced licensed phlebotomist using a 21-gauge needle. Light tourniquet pressure was applied in all subjects to assist venipuncture. Blood was collected into tubes containing anticoagulant ethylene diamine tetraaceticacid potassium (EDTA-K+) and then centrifuged for 10 min at 5,000×g to remove platelets. The plasma was then removed, aliquoted and stored at -80°C until all the samples were collected and then the oxidative parameters were tested. All blood samples were collected at approximately the same time of day when tour-inspection staff completed their inspection work, and also in case the circadian variations might exert effects on oxidative stress levels. Determination of Plasma T-AOC level
T-AOC was determined with an Ultra-spect spectrophotometer (Pharmacia, Houston, Texas, USA) following the procedure described by Benzie and Strain [Benzie et al., 1996] with modifications. A colored ferrous-TPTZ (tripyridyl-triazine) complex was generated at low pH while Fe3+ ion was reduced to Fe2+ ion, which resulted in an increase in absorbance at 593 nm. Serum proteins and low molecular weight -SH rich compounds did cause much interference, because they had very low activity in this method. Briefly,
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50 µl plasma were diluted with 50 µl deionized water and then added to 900 µl of ferric reducing antioxidant power (FRAP) reagent Sample absorbance at 593 nm was continuously measured for 10 min at 37°C. In total, 100 µl of water was added to the control (blank) samples. A calibration curve was performed for aqueous solutions of ferrous sulfate (FeSO4) (50-1200 µM). Results were expressed in μM. Determination of GPx Activity GPx activity was measured by following the GPx Assay Kit instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, the reaction mixture contained 2.7 mL of potassium phosphate buffer (0.067 M, pH 6.6), 0.1 mL EDTA (15 mM), 0.1 mL of Nicotinamide Adenine Dinucleotide Phosphate (NADPH) (6 mM in 1% [w/v] sodium bicarbonate), and 50 µL of sample. A change in color was monitored by recording absorbance (340 nm) at 1-minute intervals for 3 mins. One unit of the enzyme activity (U/min/mg) was expressed as n moles of nicotinamide adenine dinucleotide phosphate (NADPH) oxidized/min/mg protein using the molar extinction coefficient of NADPH (6.22 ×106 M−1cm−1). Measurement of Serum SOD Activity
SOD activity in the serum of all subjects was measured by the xanthine oxidase method using a SOD Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer’s instructions. The xanthine oxidase system generates a superoxide anion radical which oxidates hydroxylamine and simultaneously generates prunosus nitrite compounds in the presence of a color-developing agent. The SOD activity of each sample is in a linear correlation with absorbance, and expressed as U/ml.
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Cytokinesis-block Micronucleus Test A micronucleus test was performed referring to the standard procedure [Fenech et al., 2007], with minor modifications, after cytokinesis was blocked. Briefly, 4.7 ml of serum-free cell freezing medium 1640 (Roswell Park Memorial Institute (RPMI)-1640) containing 10% fetal bovine serum (FBS), 1% L-glutamine and antibiotics (100 IU/ml penicillin, and 100 μg/ml streptomycin) was added to 0.3 ml whole blood for lymphocytes culture. Next, 2% phytohemagglutinin (PHA) was added to stimulate cultures. Two independent cultures for each subject were incubated at 37°C, 5% CO2 and 90% humidified atmosphere for 44 hrs. Next, 6 μg/ml cytochalasin-B was added to the cultures to block cytokinesis for another 28 h (whole culture time 72 h). The cells were harvested and subjected to a hypotonic state. Cells were then prefixed with ice-cold fixative solution (5:1 v/v methanol: acetic acid), and then washed and fixed with 10 ml of fresh fixative for 1 h on ice. Cell suspensions were finally dropped onto slides and then stained for 5 min with 4% Giemsa in Sörensen’s phosphate buffer (0.067 M Na2HPO4/KH2PO4, pH 6.8) after dried. Microscopy was performed at 400× magnification and then confirmed at 1,000× magnification. All slides were analyzed blindly. The
MCF in 500 cells of each independent culture with well-preserved cytoplasm was scored and calculated as: micronucleus cell number/all the cells counted×100%, MN calculated as: micronucleus number/ all the cells counted×100%, according to established criteria. Statistical Analysis The group average of each serum oxidative stress marker and the ratio of micronucleus were calculated and presented as mean±standard deviation (SD),
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and range for 95% confidence interval was included. The total group averages and averages in different exposure durations and exposure level groups of serum oxidative stress markers were stratified, analyzed, and compared using a one-way analysis of variance (ANOVA) to determine if any statistically significant difference existed. The differences of MCF and MN between the control and exposure group were compared with a u test in Poisson Distribution. All P values less than 0.05 were considered statistically significant. All the statistical analyses were performed using SPSS 11.0 software (SPSS.Inc, Chicago, Illinois, USA).
Results The ELF-MF intensity in the route of tour-inspections nearby 500 kV and 220 kV transformers and distribution lines which were selected randomly from the 11 of the total 22 transformer stations was measured respectively. In the 500 kV area, the intensity of magnetic filed intensity at the spots ranged from 0.62 to 30.19 μT and the median ELF-MF level was 18.72 μT. The magnetic filed intensity in the 220 kV area ranged from 0.51-60.11 μT, and the median ELF-MF level was 17.72 μT. In the environment of the control, Max and Min ELF-MF levels were 0.72 and 0.07 μT respectively, and the median ELF-MF
level was 0.21 μT was much lower than that in both 220 kV and 500 kV. Individual ELF-MF exposure doses were calculated and expressed as 8 hr TWA according to the exposure time provided by the subjects’ detailed record of work days. The average TWA was 7.3 μT ( 1.56 ~ 26.33 μT ) and the subjects were subgrouped by cumulative ELF-MF exposure dose: low (< 1.56 μT ), middle ( 1.56 ~ 7.3 μT ) and high ( > 7.3 μT). Total oxidative stress level analysis
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Overall, 560 male workers were included in oxidative stress level analysis including 280 exposed individuals and 280 control individuals. The 280 male workers in the exposure group (average age 30.9±5.6) were divided into 2 sub-groups by exposure duration, 154 male workers in ＜5 y exposure group and 126 male workers in ≥5 y exposure group. There were 50 female workers (average age 29.8±4.5) in this study, including 20 females in the control group and 30 females in the exposure group. The total differences of T-AOC, SOD, MDA, GPx, MCF and MN levels between the control and exposure groups were analyzed with a u-test. The average levels of T-AOC, SOD, MDA, GPx, MCF and MN in the control vs exposure groups were respectively:15.3 μM vs 15.1 μM, 108.4 U/ml vs 107.4 U/ml, 4.5μM vs 4.3 μM, 200 nmol NADPH oxidized/min/mg protein (U/min/mg) vs 197 U/min/mg, 2.6% vs 2.8%, 2.7% vs 3.0% . No statistical difference was found (P > 0.05). Considering gender as an influence， the male workers and female workers were analyzed separately. The results showed the levels of T-AOC, SOD, MDA and Gpx between female exposure and control groups were not statistically different. The difference among the sub-groups of female workers was not analysed due to the limited number. When all the male
subjects were sub-grouped, the changes of oxidative stress biomarkers level or activity among different total service time (Control,
