The Impact of Extremely Low Frequency-Electromagnetic Fields and Light at

Night (LAN) on Estradiol (E2) levels, Oxidative Stress and DNA configuration
in Female Night Shift Workers.
Kavitha Varak1, Surender V1, Bhargava Sc2, Ahuja Yr3, Tiwari R1*
1
UGC research unit, Bhavan’s new science college, Hyderabad, Telangana State, India.
2
Department of electrical and electronic engineering, Sree Nidhi Institute of Science and
Technology, Hyderabad, Telangana State, India.
3
Genetics unit, Vasavi Medical and Research Centre, Hyderabad, Telangana State, India.
E-mail ID: rravindra_tiwari@yahoo.com
Every living being on this planet is tuned into the earth’s electromagnetic fields (EMFs) and uses
them for various purposes. Human bodies are essentially very sensitive electromagnetic systems.
Since last four decades the effects of electromagnetic fields on biological systems have been
extensively investigated. The reports are controversial and inconclusive as well. There is hardly
any study on estrogen hormone which plays a vital role in sustaining the homeostatic mechanism
pertaining to DNA integrity. The aim of the study is to assess the effect of estrogen hormone on
exposure to electromagnetic fields and light at night (LAN). Other parameters like oxidative
stress and DNA damage and DNA integrity are also included in the present study to rule out the
controversy prevailing regarding the influence of EMFs and LAN on the risk of breast cancer.
Blood samples from 400 night shift working women were collected and analyzed for estradiol
hormone level By Chemiluminescence Immunoassay (CLIA) method. DNA damage was studied
in exposed and control subjects using Single Cell Gell Electrophoresis (Scge). Oxidative Stress
was estimated by measuring levels of plasma Malonyl Dialdehyde (MDA), serum Nitric Oxide
(NO) and Chromosomal aberrations by Micronuleui Test (MNT). The analysis showed
significant increase (p<0.0001) In Estradiol hormone level in exposed, when compared to
controls. There was a significant increase in DNA damage (p<0.05). The plasma MDA levels
also demonstrated the same observation. Our findings lead us to summarize that electromagnetic
fields and light at night (LAN) elevated the estrogen levels, which suggest that these increased
levels and the DNA and oxidative stress could possibly be the risk factors in urbanized night
shift female workers.
Key Words: EMFs, LAN, Estradiol, CLIA, DNA Configuration, Oxidative Stress and MNT
INTRODUCTION:
The twenty-first century is marked with aggressive development of the wireless communications
like satellites, mobile phones, Internet, Wi-Fi, computers etc. It is fair to say that that the entire
civilization, both biosphere and mankind are exposed to continuous exposure of EMFs and the
number of persons exposed to EMFs is still growing [1]. Recently, the effects of extremely low-
frequency electromagnetic fields (ELF EMF) on biological systems have been extensively
investigated. The biological effects of EMFs are among growing concerns which have remained
largely unanswered [2].
Smartphones are now owned by most teenagers and adults in many countries [3]. Scientific
research into possible health effects of ELF-EMFs on women also indicated conflicting results
and is inconclusive [4]. Recent studies on EMFs have shown considerable effect on endocrine
system, particularly stress hormones and gonadal hormones of females [5][6][7]. The possible
association between ELF-EMFs, leukemia and breast cancer has been discussed widely since
1979 [8], [9],[10],[11] particularly when the epidemiological study suggested a possible link
between ELF-EMFs and childhood leukemia. Further it is to be noted that International
Association on Research in Cancer (IARC) under aegis of the World Health Organization
(WHO) has classified ELFs as a Possible Human Carcinogen in the Group 2B category [12].
ELF-EMFs, Light at Night and Estradiol:
Melatonin levels are believed to be inversely related to estrogen levels. For example, when
melatonin levels are low, estrogen levels are believed to be high, and vice-versa [13]. Therefore,
if ELF – EMFs and LAN suppress the normal nocturnal rise in melatonin, estrogen levels would
subsequently be increased. Because increased levels of estrogen are hypothesized to increase the
risk of breast cancer, the suppression of melatonin by magnetic fields possibly could increase the
risk of breast cancer. [11], [14], [15], [16], [17].
Melatonin's direct effect on mammary tumor cells is that it interferes with the activation of the
estrogen receptor, regulates the activity of the aromatases, the enzymes responsible for the local
synthesis of estrogens, thus behaving as a selective estrogen enzyme modulator. The same
molecule has both properties to selectively neutralize the effects of estrogens on the breast and
the local biosynthesis of estrogens from androgens [18].
 Fig 1. Diagram showing the relation between
Melatonin and Estrogen receptors: (a) indirect
effects on the neuroendocrine reproductive axis;
(b) anti-estrogenic actions on the breast cancer
cells, melatonin thus behaving as a selective
estrogen receptor modulator (SERM) and a
selective estrogen enzyme modulator (SEEM); (c)
anti-oxidative effects; (d) immune modulatory
actions; or (e) inhibition of telomerase activity

Estrogen levels may be implicated in breast cancer risk because of its role in stimulating breast
cell division during the critical periods of breast growth and development [19], [20], [21], [22].
A woman's risk for breast cancer is associated with increased levels of estrogen. It is important
to understand how lifestyle and environmental factors (ELF-EMFs and LAN) may affect the
increased levels of estrogen [23].
MATERIALS AND METHODS:
The women (n=400) working in the night shifts for about 5 years, from different hospitals and
software companies situated in various locations in Hyderabad, India, were considered for the
study.
Proforma/Questionnaire
Age, gender, diet, exercise and recent medical history were used as criteria for the selection of
both exposed and control populations. Detailed questionnaire on work characteristics,
psychologically perceived stress and non-specific symptoms experienced by exposed populations
were responded by 94% of participants. Questionnaire on subjective symptoms related to EMF
exposure included self assessment of non-specific symptoms such as headache, dizziness,
tinnitus, visual impairments, sleep disturbances and menstrual cycle.
Sampling
After receiving informed consent, 5 ml peripheral blood was collected in the early hours between
6 am to 8 am from each volunteer by venepuncture into a sterilized disposable syringe. 1ml of
whole blood was allowed to clot to collect the serum. The remaining 4 ml of blood was
heparinized and both the samples placed in ice to prevent exogenous damage. The samples were
processed for estimating Estradiol by CLIA, DNA damage by SCGE, oxidative stress by LPO
and NO and Chromosomal Aberrations by MNT- Micronuclei Test.
Estradiol assay- Chemiluminescence Assay (CLIA)
Plasma Estradiol was quantitatively measured by Chemiluminescence Assay (CLIA) [24]. The
E2 EIA is based on the principle of competitive binding between E2 in the test specimen and E2-
HRP conjugate for a constant amount of rabbit anti-Estradiol. In the incubation, goat anti-rabbit
IgG coated wells are incubated with 25 μl E2 standards, controls, samples, 100 μl Estradiol-
HRP Conjugate Reagent and 50 μl rabbit anti-Estradiol reagent at room temperature for 90
minutes. A solution of chemiluminescent substrate is then added and read Relative Light Units
(RLU) with a Luminometer. The intensity of the emitting light is proportional to the amount of
enzyme present and is inversely related to the amount of unlabeled E2 in the sample.
Quantification of unknown samples were achieved by comparing their activity with a reference
curve prepared with known calibrators.
SCGE - Alkaline Comet assay
A sensitive test to study the degree of DNA damage was used in this study. [25],[26] The cells
from each sample were mixed with low melting point agarose and embedded on a layer of
normal melting point agarose, precoated on a micro slide. On top, a third layer of low melting
point agarose was spread. The slides were treated overnight in freshly prepared, chilled lysing
solution containing high salt concentration and detergents. DMSO was added before use and pH
adjusted to 10 at 40ºC. The slides were removed from the lysing solution, and kept in alkaline
electrophoresis unit for 30 minutes. On electrophoresis fragmented DNA produced by single
strand breaks, moved towards anode to form characteristic comets. The slides were neutralized
with Tris buffer and rinsed with distilled water. All of these steps were conducted in dim light to
prevent additional DNA damage. The slides were air-dried and stored. Before staining, they were
left in fixing solution for 10 minutes, washed several times with water and dried thoroughly.
Silver staining method was used to visualize the nature of DNA. [27] The slides will be screened
under a bright field transmission microscope. Comet tail length, which is an estimate of DNA
damage was measured for 100 cells per treatment using ocular micrometer.
Lipid Peroxidation - Thiobarbituric Acid Reactive Species (TBARS) Assay
Malondialdehyde [MDA] is the stable end product of lipid peroxidation and an efficient
parameter for lipid oxidative damage. [28] Formation of MDA is assessed by quantification of
thiobarbituric acid reactive species (TBARS). The protein free plasma obtained after
centrifugation was treated with thiobarbituric acid (0.33%) in boiling water bath for 30minutes to
form an 1:2 adduct which was measured at 535 nm with spectrophotometer (Schimadzu UV-
160A, Cat No:204-04550-01, Made in Japan). MDA standards were prepared by using 1, 1, 3, 3-
tetraethoxy propane. The concentration of MDA equivalents in nmol/ml in samples were
interpolated from the standard curve.
Estimation of Serum Nitric Oxide
Nitric Oxide (NO) is an unstable compound, it is rapidly converted to nitrates and nitrites in the
body, hence their concentration is parallel to NO levels. Total production of NO was determined
by assaying for nitrite and nitrate. Nitrate and nitrite concentrations were then estimated by the
Griess method. [29] In this method nitrate is first reduced to nitrite which is treated with
sulfanilamide and N1-napthyl-ethylene diamine. The coloured compound is formed after which
its characteristic absorption spectrum was determined. The optical densities of the samples were
then obtained on a spectrophotometer at 540 nm (Schimadzu UV-160A, Cat No:204-04550-01,
Made in Japan).
Chromosomal Aberrations - MNT- Micronucleui Test:
Micronuclei (MN) were observed in cytokinesis blocked cells using cytochalasin B (Cyt-B)
following the method suggested by Fenech and Morley [30]. About 0.2 mL of PHA was added to
5 mL of RPMI 1640 medium using 1 mL syringe. 15 drops of blood was added to each vial.
Samples were initiated in duplicates. The culture vials were incubated for 72 h at 37 °C and
shaken for proper mixing. Cyt-B (6 g/mL) was added at 44th hour after initiation of culture and
incubated further for another 28 h at 37 °C and then cultures were harvested. The cultures were
centrifuged at 1000 rpm for 5-10 min. The supernatant was discarded and 5 mL of prewarmed
hypotonic solution (0.56%) was added to the pellet drop by drop slowly on by vortexing and
incubated for 10 min for 37 °C. Then the vials were centrifuged for a minute and supernatant was
discarded. Cells were fixed in 5 mL of fixative (3:1 methanol: acetic acid) followed by two more
changes of fixative. The slides were prepared in triplicate by gently dropping the cell suspension
onto the precleaned slides and flame dried. Slides were stained with 2% Giemsa for 10 min,
rinsed and air dried. To determine the MN yield, nearly 1000 binucleated (BN) cells were scored
for each experimental condition under magnifications of 400x and finally 1000x from 2 coded
slides/culture. Identification of MN was according to the criteria summarized by Countryman
and Heddle [31]. Routinely on average ~2000 BN cells were scored for the presence of MN in
each subject and mean values of the results were calculated.
STATISTICAL ANALYSIS:
The slides were coded during processing and decoded at the time of statistical analysis. For
statistical evaluation, observations on each parameter for each group were pooled and mean ±
SD (SE), Student’s t-test (paired and unpaired comparisons), one way Analysis of Variance
(ANOVA) were calculated.
RESULTS:
In this study the exposure effect of ELF-EMFs and LAN on Estradiol levels, DNA integrity,
Oxidative stress and Micronuclei from 400 subjects was studied. The t-value for Estradiol
estimation was found to be 22.99, the p-value was <00001. The result was significant at p < .05.
The alkaline comet assay indicated DNA damage ranging between 1.72µm to 9.23µm. The
maximum tail length (9.923 + 0.67µm) was observed in night shift female workers. The mean
MDA concentrations (nmoles/ml) showed increase in MDA levels when compared with controls.
The levels of serum nitric oxide, determined as nitrites or nitrates showed an increased level of
NO in the night shift workers (p=0.033). A significant stepwise increase was found in the
percentage of micronuclei from control to subjects.
Estradiol levels (pg/ml)
Subjects Mean + SD Range T value p value
130.56-
EMF-Exposed 400 284.4 + 89.08 736.27 <
Control 200 72.10 + 34.08 33.7 - 169 22.99 .00001
DNA Damage Length (arbitrary units)
Subjects N Mean + SD Range Variance
EMF-Exposed 400 8.76 + 1.31 1.72 - 9.23 1.723
Control 200 4.30 + 1.13 1.47-7.49 1.287
MDA Levels (nmoles/ml)
Subjects N Mean + SD Range T value P value
EMF-Exposed 400 2.67 + 0.82 0.90 - 9.41
Control 200 1. 87 + 1.47 0.94 - 5.23 2.045 0.042
NO Levels (µmoles/ml)
Subjects Mean + SD Range T value p value
EMF-Exposed 400 2.25 ± 0.09 0.42 - 4.58
Control 200 2.00 ± 0.07 0.66 - 4.30 2.148 0.033
% of Micronuclei
Subjects N Mean + SE
EMF-Exposed 400 1.39 + 0.63
Control 200 1.24 + 0.67
Table1. Results of DNA Damage, MDA, Serum Nitric Oxide, Estradiol levels and MNT in
the exposed compared with control group
 40
35
30
25
Age

20
15
10
5
0
Group 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800
Estradiol concentration (pg/ml)
exposed

Fig2. Graph showing Estradiol E2 (pg/ml) concentration Fig3. SCGE- Comet Tails
CONCLUSION AND DISCUSSION:
The so far results suggested the induction of Elevated Levels of Estradiol (E2) in Exposed
compared to controls suggests to rule out the possibility of Breast Cancer. DNA damage and
increase in frequency of micronuclei in night shift workers may be due to genotoxic action of
ELF-EMFs and LAN. The MDA levels in plasma and NO levels in serum of exposed subjects
from controls suggest a statistical significance. Thus Oxidative Stress markers could have an
important implication in the comprehension of the biological stress response to ELF-EMFs and
LAN.
With industrial revolution, our exposure to EMFs, even at power line frequencies, has increased
considerably. There is every possibility of synergistic effects of EMFs with other pollutants.
Biological systems are highly complex and it is unlikely that a single mechanism could explain
the effects of EMFs on them. It is reasonable to expect that more than one interaction may be
responsible for the biological effects.
Given the ubiquitous nature of EMFs, their widespread applications, and their capability to
produce deleterious effects, conclusive investigations of the health risks are critical. With the
published literature on EMF, it is still not sufficient enough to reach to a concrete conclusion.
But the possibility of negative consequences cannot be excluded. Several studies with
appropriate methodologies reflect the capacity of EMFs to cause adverse health effects. There is
a need for standardized research methodology along with the inclusion of appropriate exposure
assessment technique which is crucial for identification of dose response relation if any and the
elucidation of mechanism for biological interaction. If we do not work upon the demerits of
previous findings, we may remain far from any concrete conclusion. At the same time, it is
critical to analyze the EMF investigations giving more weight to the similarities and
dissimilarities rather than giving more importance to the endpoints reached. For the time being,
since it is difficult to protect oneself from EMFs, the only practical way to check exposures is to
distance oneself from the source. Together, the precautionary approach and ALARA (As Low As
Reasonably Achievable) principle can also be applied to save us from substantial exposures and
the possible ill effects if any. Nonetheless, we reckon that the underlying mechanisms of
interaction between EMF and the estradiol hormone are still to be completely understood and
need further studies at a molecular level.
We strongly advocate that with mere swelling number of studies no fruitful conclusions can be
reached. If we do not address the limitations of past investigations, we may not be able to truly
contribute to the domain of bioelectromagnetics. Therefore, the need of the hour is to do
innovative research with sound designs and appropriate methodologies rising above the demerits
of past studies.
A preliminary warning has been already given by the Working Group of the National Institute of
Environmental Health Sciences of the USA (NIEHS) and The International Agency for Research
on Cancer (IARC). A part of World health organization (WHO) has concluded that extremely
low frequency EMF is a class 2B (possible) human carcinogen [12].
Further research using standardized methodology is essential with active support from all
concerned, viz. industry, government and philanthropic organizations. Only such an effort can
help in formulating suitable guidelines for the use of EMFs. A line should be drawn between use
and abuse until a “clean chit” is issued.
REFERENCES:
1. Markov M, Grigoriev Y. “Protect children from EMF”. Electromagn Biol Med. 2015
Sep;34(3):251-6.
2. Shahbazi-Gahrouei D, Hashemi-Beni B, Ahmadi Z. “Effects of RF-EMF Exposure from
GSM Mobile Phones on Proliferation Rate of Human Adipose-derived Stem Cells: An In-
vitro Study”. J Biomed Phys Eng. 2016 Dec 1;6(4):243-252.
3. Redmayne M. “Where's Your Phone? A Survey of Where Women Aged 15-40 Carry Their
Smartphone and Related Risk Perception: A Survey and Pilot Study”. PLoS One. 2017 Jan
6:12(1):e0167996
4. Vijayalaxmi, Obe G. “Controversial cytogenetic observations in mammalian somatic cells
exposed to radiofrequency radiation”. Radiat Res 2004; 162(5):481-96.
5. Hong ME, Yoon KH, Jung YY, Lee TJ, Park ES, Sohn UD, et al. Influence of exposure to
extremely low frequency magnetic field on neuroendocrine cells and hormones in stomach
of rats. Korean J Physiol Pharmacol 2011; 15:137-142.

6. Seyed Mohammad Mahdavi, Hedayat Sahraei, Parichehreh Yaghmaei, Hassan Tavakoli.

“Effects of Electromagnetic Radiation Exposure on Stress-Related Behaviors and Stress
Hormones in Male Wistar Rats”. Biomol Ther (Seoul). 2014 Nov; 22(6): 570–576.
7. Ali Asghari, Amir Afshin Khaki, Asghar Rajabzadeh, and Arash Khaki. “A review on
Electromagnetic fields (EMFs) and the reproductive system”. Electron Physician. 2016 Jul;
8(7): 2655–2662.

8. Wertheimer N, Leeper E. “Electrical wiring configurations and childhood cancer”. Am J

Epidemiol 1979; 109(3):273-84.
9. Schernhammer ES, Landent F, Speizel FE, Willet, WC, Hunta DJ, Kawachi L, et al.
“Rotating night shifts and risk of breast cancer in women participating in the nurses health
study”. J Natl Cancer Inst 2001;93:1563-8.
10. Greaves M. “Childhood leukaemia”. BMJ 2002; 324:283-7.
11. Daniel Luzer."Night Shift Work and Breast Cancer Risk".J Natl Cancer Inst 2016;108 (12):
djw241
12. BioInitiative. “A Rationale for Biologically-based Exposure Standards for Low-Intensity
Electromagnetic Radiation”. 2012; Bioinitiative Report;Pg:65.
13. Maganhin CC1, Carbonel AA, Hatty JH, Fuchs LF, Oliveira-Júnior IS, Simões Mde J,
Simões RS, Baracat EC, Soares-Jr JM. “Melatonin effects on the female genital system: a
brief review”. Rev Assoc Med Bras 2008; 54(3):267-71.
14. Blask DE, Brainard GC, Dauchy RT, Hanifin JP, Davidson LK, Krause JA, et al.
“Melatonin-depleted blood from premenopausal women exposed to light at night stimulates
growth of human breast cancer xenografts in nude rats”. Cancer Res 2005; 65:11174-84.
15. Ravindra T, Lakshmi NK, Ahuja YR. “Melatonin in pathogenesis and therapy of cancer”.
2006 Indian J Med Sci 60 (12) 523-535
16. Brainard GC, Kavet R, Kheifets LI. “The relationship between electromagnetic field and
light exposures to melatonin and breast cancer risk: a review of the relevant literature”. J
Pineal Res. 1999; 26(2):65-100
17. Merklinger-Gruchala A, Ellison PT, Lipson SF, Thune I, Jasienska G. “Low estradiol levels
in women of reproductive age having low sleep variation”. Eur J Cancer Prev 2008;
17(5):467-72.
18. Samuel Cos MD, PhD*, Alicia Gonza´lez PhD, Carlos Martı´nez-Campa PhD, Maria
Dolores Mediavill'a MD, PhD, Carolina Alonso-Gonza´lez BS, Emilio J. Sa´nchez-Barcelo´
MD, PhD. “ Estrogen-signaling pathway: A link between breast cancer and melatonin
oncostatic actions”. Cancer Detection and Prevention 2006; 30: 118–128.
19. Parl FF1, Dawling S, Roodi N, Crooke PS."Estrogen metabolism and breast cancer: a risk
model".Ann N Y Acad Sci. 2009;1155:68-75.
20. Rodriguez C1, Patel AV, Calle EE, Jacob EJ, Thun MJ."Estrogen replacement therapy and
ovarian cancer mortality in a large prospective study of US women". JAMA. 2001;
21;285(11):1460-5.
21. Manole D1, Schildknecht B, Gosnell B, Adams E, Derwahl M."Estrogen promotes growth
of human thyroid tumor cells by different molecular mechanisms".J Clin Endocrinol Metab.
2001; 86(3):1072-7.
22. Yager JD."Endogenous estrogens as carcinogens through metabolic activation".J Natl
Cancer Inst Monogr. 2000;(27):67-73.
23. Girgert R, Gru¨ndker C, Emons G, Hanf V. “Electromagnetic fields alter the expression of
estrogen receptor cofactors in breast cancer cells”. Bioelectromagnetics 2008; 29:169–76.
24. Ratcliffe, W.A., Carter, G.D., Dowsett, M., et al., Oestradiol assays: applications and
guidelines for the provision of a clinical biochemistry service, Ann. Clin. Biochem., 1988;
25:466-483.
25. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low
levels of DNA damage in individual cells. Experimental Cell Research 1988; 17:184–191.
26. McGlynn, A.P., Wasson, G.R., O’Connor, J., McKerr, G., McKelvey-Martin, V.J., &
Downes, C.S. (1999). The Bromodeoxyuridine Comet Assay Detection of Maturation of
Recently Replicated DNA in Individual Cells. Cancer Res, 59, 5912 – 5916.
27. Ahuja, Y.R., & Saran, R. (1999). Alkaline single cell gel electrophoresis assay/comet assay
protocol. Journal of Cytology and Genetics, 34, 57–62.
28. Halliwell, B., & Gutteridge, J.M.C. (1999). Free radicals, other reactive species, and
disease. Free Radicals in Biology and Medicine, 3rd Ed. Oxford University Press, Oxford.
29. Stuehr, D.J., Gross, S.S., Sakuma, I., Levi, R., & Nathan, C.F.(1989). Actived murine
macrophages secreted a metabolite of arginine with the bioactivity of the endothelium-
derived relaxing factor and the chemical reactivity of nitric oxide. Journal of Experimental
Medicine, 169 (3), 1011-1023.
30. Fenech M, Morley. “Measurement of micronuclei in lymphocytes”. AA Mutat Res. 1985
Feb-Apr;147(1-2):29-36.
31. Countryman PI, Heddle JA. “The production of micronuclei from chromosome aberrations
in irradiated cultures of human lymphocytes”. Mutat Res. 1976 Dec; 41(2-3):321-32.