A study protocol for investigating the effects of mobile phone-originated electromagnetic waves on thyroid gland and thyroid hormone activities in the brain

Hendra Zufry; Achmad Rudijanto; Djoko Wahono Soeatmadji; Setyawan Purnomo Sakti; Khairul Munadi; Hidayat Sujuti; Karyono Mintaroem

doi:10.12688/f1000research.129735.1

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Study Protocol

A study protocol for investigating the effects of mobile phone-originated electromagnetic waves on thyroid gland and thyroid hormone activities in the brain

[version 1; peer review: 2 approved with reservations]

Hendra Zufry ^1,2, Achmad Rudijanto³, Djoko Wahono Soeatmadji⁴, [...] Setyawan Purnomo Sakti⁵, Khairul Munadi⁶, Hidayat Sujuti⁷, Karyono Mintaroem⁸

Hendra Zufry ^1,2, Achmad Rudijanto³, [...] Djoko Wahono Soeatmadji⁴, Setyawan Purnomo Sakti⁵, Khairul Munadi⁶, Hidayat Sujuti⁷, Karyono Mintaroem⁸

PUBLISHED 03 Feb 2023

Author details Author details

¹ Doctoral Program in Medical Sciences, Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia
² Division of Endocrinology, Metabolism, and Diabetes, Thyroid Center, Department of Internal Medicine, Universitas Syiah Kuala, Banda Aceh, 23111, Indonesia
³ Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia
⁴ Division of Endocrinology and Metabolic Diseases, Department of Internal Medicine, Universitas Brawijaya, Malang, 65145, Indonesia
⁵ Department of Physics, Faculty of Mathematics and Natural Sciences, Universitas Brawijaya, Malang, 65145, Indonesia
⁶ Department of Electrical and Computer Engineering, Faculty of Engineering, Universitas Syiah Kuala, Banda Aceh, 23111, Indonesia
⁷ Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia
⁸ Department of Pathological Anatomy, Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia

Hendra Zufry
Roles: Conceptualization, Funding Acquisition, Investigation, Methodology, Resources, Visualization, Writing – Original Draft Preparation

Achmad Rudijanto
Roles: Supervision, Validation, Writing – Review & Editing

Djoko Wahono Soeatmadji
Roles: Supervision, Validation, Writing – Review & Editing

Setyawan Purnomo Sakti
Roles: Supervision, Validation, Writing – Review & Editing

Khairul Munadi
Roles: Supervision, Validation, Writing – Review & Editing

Hidayat Sujuti
Roles: Supervision, Validation, Writing – Review & Editing

Karyono Mintaroem
Roles: Supervision, Validation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

With widespread users across the globe, topic on the impact of mobile phone in particular regarding its electromagnetic wave radiation deserves a priority. Carcinogenic effects of the electromagnetic wave radiation have been suggested by multiple studies. Effects of the radiation are to involve thermal and non-thermal pathways. Among human organs, thyroid gland is susceptible to the radiation exposure due to its location being superficial and close to the radiation transmitter. Previous studies have indicated the correlation between the mobile phone radiation and thyroid dysfunctions. However, a conclusion that the electromagnetic wave radiation is the causative agent of the thyroid dysfunctions has not been reached. Most of the study are unable to remove the influence of the external electromagnetic wave radiation. Hence, this study aimed to perform an investigation with true experimental design using animal model in an anechoic chamber – an isolated chamber which could minimize the electromagnetic wave noise from the outside. In this article, we present the description of the anechoic chamber along with the research protocols. Parameters affecting the investigation, including signal frequencies inside the chamber and specific absorption rate are also measured.

Keywords

Carcinogenicity; cellular phone; radio frequency; thyroid stimulating hormone; thyroxine

Corresponding author: Hendra Zufry

Competing interests: No competing interests were disclosed.

Grant information: The author(s) declared that no grants were involved in supporting this work.

Copyright: © 2023 Zufry H et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Zufry H, Rudijanto A, Soeatmadji DW et al. A study protocol for investigating the effects of mobile phone-originated electromagnetic waves on thyroid gland and thyroid hormone activities in the brain [version 1; peer review: 2 approved with reservations]. F1000Research 2023, 12:132 (https://doi.org/10.12688/f1000research.129735.1) First published: 03 Feb 2023, 12:132 (https://doi.org/10.12688/f1000research.129735.1) Latest published: 09 May 2023, 12:132 (https://doi.org/10.12688/f1000research.129735.2)

Introduction

Mobile phone is the most used electronic devices working based on the electromagnetic radio frequency. According to the Oceania Radiofrequency Scientific Advisory Association (ORSAA), the cellular phone usage has reached seven billion individuals or 97% of total world’s population.¹ In East Asia, the number of mobile phone users is twice as many as its own population (600 million individuals).² The usage duration ranged widely across countries, from 25 minutes in Japan up to 248 minutes in the Philippines.³ In Switzerland, an individual could spend 30 minutes on their phone.³ In the midst of hyperconnected world, people become more urged of using their phone which is done in at least every 30 minutes.⁴ With such numbers, the assessment of the effects of mobile phone radiation on human health are urgently needed.

An epidemiological studies have revealed that 30 min daily use of mobile phone lasted for more than 10 years could increase the risk of glioma in brain cells.⁵^,⁶ Short-term exposure of the radiation has been reported to be associated with the increased cellular immune activities.⁷^,⁸ Meanwhile, its long-term exposure could cause irreversible cellular damage.⁹ Impacts of phone usage is associated with the non-ionic electromagnetic wave radiation transmitted when making or receiving a call.¹⁰

In general, there are two main pathways explaining the biological dysfunctions caused by the exposure of mobile phone-derived electromagnetic wave. The first is through the thermal effects generated by the heat radiation transmitted by the electromagnetic wave, while the latter is through non-thermal effects which have not been completely understood.¹¹ Carcinogenicity of phone radiation has been reported to be associated with increased Ca²⁺ efflux, oxidative stress imbalance, DNA damage, elevation of ornithine decarboxylase and protein kinases activities, disrupted activity of Na⁺/K⁺ phosphatase, melatonin level reduction, disruption in histone kinase activity, and errors in protein synthesis.¹²^–¹⁴ Multiple reports have established a correlation between the electromagnetic radiation and impaired estrogen production.¹⁵^–¹⁷ Estrogen shares a similarity with thyroid hormones of being active in nuclear receptors, hence could be affected by the phone radiation in a similar way.¹⁸

Thyroid gland, a most important endocrine gland, is the most susceptible to the effect of electromagnetic wave radiation from cellular phone owing to its superficial location and close distance with the cellular phone when being used.¹⁹^,²⁰ A systematic review revealed various effects of the radiation on thyroid function and level of serum thyroid stimulating hormone (TSH).²¹ Collective evidence by another systematic review also suggested changed levels of triiodothyronine (T₃), thyroxine (T₄), and TSH occurred in groups receiving cell phone radiation.¹⁹ Expression of thyroid-related biomolecules and oxidative stress in rats were found to be significantly correlated with mobile phone-originated electromagnetic radiation.²² A study suggested the significant effect of an 1800 MHz electromagnetic field might take place in hypothalamo-pituitary-thyroid axis.²³ By using a Swiss albino mice model, a study reported that the 500–900 MHz electromagnetic field was responsible for the significant reduction of serum thyroid stimulating hormone (TSH). Sub-chronic exposure of 915 MHz (8 h/day for 16 weeks) on Male Sprague–Dawley rats was found to not significantly affect serum T₃, T₄, and TSH levels.²⁴

Different and non-standardized protocols used in investigating the aforementioned effects have made scientist difficult to reach a conclusive finding. Of which, previous studies were unable to exclude the effects of non-ionizing electromagnetic radiation from outside the system. Indeed, a cage modification has been proposed in a study using anechoic chamber allowing whole-body exposure of the electromagnetic field.²⁴ Nonetheless, the possible bias from the external electromagnetic wave influence persists. Therefore, we reported the use of anechoic chamber as a mean to prevent the influence of the external radiation during the investigation.

Experimental design

This study employs a true experimental design by measuring the change of TSH and T₄ of the animal subject (Rattus norvegicus) before and after being exposed with mobile phone-originated electromagnetic wave radiation in anechoic chamber. Malondialdehyde (MDA) would also be determined before and after the exposure while nuclear receptor subfamily 3 group C member 1 (NR3C1), monocarboxylate transporter 8 (MCT8), pyruvate kinase, plasma membrane Ca²⁺ ATPase (PMCA), and thyroid hormone receptor alpha (THRA) would be determined after the exposure. In addition, the histopathological analysis on the thyroid gland will be carried out. All the results would be compared with control group (receiving no radiation exposure). The objective is to reveal the effect of mobile phone-originated electromagnetic wave radiation on thyroid function with minimum influence from the outside environment. The workflow diagram of this study has been presented in Figure 1.

Figure 1. Study workflow for investigating the effects of mobile phone-originated electromagnetic wave radiation on thyroid functions.

Thyroid stimulating hormone TSH; thyroxine, T₄; malondialdehyde, MDA; nuclear receptor subfamily 3 group C member 1, NR3C1; monocarboxylate transporter 8, MCT8; pyruvate kinase, plasma membrane Ca2+ ATPase, PMCA; thyroid hormone receptor alpha, THRA.

Equipment

• Analytical balance
• TSH and T₄ measuring kit (test tube and its rack, micropipette (200 μ), yellow tip, Vidas^®)
• Vacutainer red top
• Minor set
• Slide superfrost (Thermoscientific)
• Global System for Mobile Phone (GSM) Sim 900L
• Arduino uno microcontroller
• Real time clock (RTC) module
• Sender—receiver cellular phone module
• Logger data module
• Temperature sensor dht-22
• Spectrum analyzer

Animal

The animals are male rats (Rattus norvegicus) obtained from Faculty of Veterinary, Universitas Syiah Kuala, Banda Aceh, Indonesia. The animals must be three–four months old, weighed 160–250 g, and not showing any abnormalities.

Anechoic chamber design

Location and chamber design

Anechoic chamber has been built at Acoustic, Vibration, and Thermal Laboratory, Faculty of Engineering, Universitas Syiah Kuala, Indonesia. Installation of sound-insulating foam panels on the wall results in a chamber with field-free and sound propagation aligned with ‘inverse square law’. Specific construction design, including the access door and ventilation, allows high sound attenuation and free from structure-borne noise. The chamber has 7.8 m length, 6.3 m width, and 5 m height. It is built with two layers of wall and three layers of iron door to minimize the entrance of signal from the outside. Its interior has a dimension of 6 m × 4.5 m × 3.1 m (length, width, and height, respectively), where the airflow is maintained by a controllable ventilation. The chamber has been installed with a lighting system (6 × 80 Watt lamps) with brightness level of 500 lux. Appearance of the anechoic chamber has been presented in Figure 2.

Figure 2. Photograph of the anechoic chamber in Universitas Syiah Kuala, Indonesia.

Environmental conditions

As many as three modules of sensor dht-22 are installed surrounding the chamber interior. The sensor is connected to a microcontroller using cable to record the temperature and humidity. Data obtained in the microcontroller are then recorded and displayed through a computer.

Signal strength

Signal strengths from the Global System for Mobile communication (GSM) in the anechoic chamber are measured with spectrum analyzer (max. 3000 MHz). The schematic diagram for signal strength measurement using spectrum analyzer has been presented in Figure 3.

Figure 3. Equipment set-up for signal strength measurement.

Source of radio frequency

The signal of radio frequency (RF) in the anechoic chamber has a frequency of 891—893 MHz (GSM and UMTS signals) with Gaussian Minimum-Shift Keying (GMSK) modulation scheme for GSM, Quadrature Phase Shift Keying (QPSK) – UMTS, and 8PSK – EDGE. In the case of GMSK, the signal is smoothed using low-pass Gaussian filter prior to its modulation into signal carrier and proceeding to frequency modulator. As for QPSK and 8PSK, variants of Phase Shift Keying (PSK), the difference is only located at the code rate capacity. The design and photographed images of the GSM module used in this research have been presented in Figure 4.

Figure 4. Design (left) and photographed images (right) of the GSM signal sender (a) and receiver (b).

Determination of noise and radiation frequency

The signal noise in the anechoic chamber is very low, ranged from -79 dBM to -74 dBM (1.2589 × 10^-8 mW). The number was obtained from the determination using spectrum analyzer. Several measurement results of the RF/GSM signal frequencies at distance of 10–50 cm has been presented in Table 1.

Table 1. Results of the measurements of signal frequencies from RF/GSM inside the anechoic chamber.

Distance (cm)	mW	dBM
10	0.00005011	-43.83
20	0.00005011	-43.35
30	0.3981	-4.61
40	0.05011	-13.73
50	0.0003162	-35.89

Design and installation of RF applicator

RF applicator, in a form of a cage, has a dimensional size of 33 × 20 × 7 cm³ with pentagonal shape divided into five chambers. The cage is made from glass where wire mesh is used to cover the top for air circulation during experiment. The distance between the RF signal and the cage is maintained at 10–30 cm.

Determination of specific absorption rate

The level of electromagnetic wave radiation is represented by Specific Absorption Rate (SAR), an expression to explain the absorbance rate of the electromagnetic radiation (RF) per time unit per kilogram (W/kg). SAR is varied in different parts of the body. The mathematical expression of SAR has been presented in Equation 1.

(1)

SAR = \frac{σ E^{2}}{ρ}

Where $σ$ is electrical conductivity of the body tissue (S/m), $E$ is electrical field (V/m), and $ρ$ is the density of body tissue (kg/m³).

In the real world, the value of $ρ$ is varied, considering each rat has differences in their skin, fat, bones, and so on. Herein, the body density of each rat is assumed to be 1000 kg/m³ with $σ$ = 1 S/m for 2 GHz frequency. The value is applicable for rat with the size of around 20 cm × 4.5 cm × 4.5 cm with an averaged body weight of around 200 g.

To calculate SARS value, it is of importance to calculate the depth of RF signal penetration using the following equation (Equation 2):

(2)

δ = \sqrt{\frac{1}{π μ σ f}}

Where $δ$ is the depth of RF penetration (m), $μ$ is tissue permeability (H/m), and $f$ is averaged RF signal frequencies (mW). In the case of RF penetration into throat tissue, the $δ$ is 1.1 and the averaged uplink frequency for GSM is 9902.5 MHz. Therefore, by using the Equation 2 we obtain the value for $δ$ = 0.0159 m.

In a GSM 900 system the maximum power is 2 W; hence, the maximum averaged power ( $P$ ) is 250 mW (1/8 maximum power). If the averaged distance between the phone antenna and rat ( $A$ ) is 10 cm, the radiation density with maximal penetration ( $I$ ) could be calculated using Equation 3 resulting in $I$ = 1.481 W/m². Electrical current is expressed by Equation 4 which can be derived to Equation 5 by subtituting $P$ . With the skin impedance ( $η$ ) value of 59.094 Ohm, the electrical field strength ( $E$ ) could be calculated using Equation 5 resulting in $E$ = 9.355 V/m. By inserting all the values required in Equation 1, the SAR is obtained to be 0.0962 W/kg.

(3)

I = \frac{P}{A}

(4)

P = \frac{E^{2}}{η}

(5)

E = \sqrt{I x η}

Procedure

Randomization and acclimation

Simple random sampling is employed to randomly assign the animals into four groups, where each group consists of 10 animals. All subjects are considered having the same conditions prior to the treatment. The group assignment has been presented in Table 2.

Table 2. Group assignment.

Group	Treatment
Group I (n=10)	Fed ad libitum. Receiving no electromagnetic wave radiation.
Group II (n=10)	Fed ad libitum. Exposed with electromagnetic wave radiation with 4G frequency for 120 min/day.
Group III (n=10)	Fed ad libitum. Exposed with electromagnetic wave radiation with 4G frequency for 150 min/day.
Group IV (n=10)	Fed ad libitum. Exposed with electromagnetic wave radiation with 4G frequency for 180 min/day.

Adaptation

The animals, aged two–three months with body weights ranged from 160 to 200 g, are acclimated at room temperature in 12 h dark–12 h light cycle. The animals are fed ad libitum with standardized pellets and water, while being contained in a plastic cage (40 × 25 × 20 cm). The acclimation process lasts for seven days with observation on their behaviors and physical conditions.

Intervention

Rats in intervention group (Group II–IV) are exposed with mobile phone-derived electromagnetic wave radiation for 120, 150, and 180 min/day, respectively. The radiation was exposed in dark condition using two bidirectionally interacting GSM modules, in which their set-up has been presented in Figure 5. RTC is used to control the time of the interaction. The radiation is produced from the stimulation of GSM multiband 4G LTE signal with a frequency of 1800 MHz (Figure 6). The exposure is carried out for 12 consecutive weeks in an anechoic chamber.

Figure 5. Photographed images of the empty RF applicator taken from the top view (a). The side view of the RF applicator installation inside the anechoic chamber while being used for the radiation exposure (b). The cage is equipped with three dht-22 temperature sensors modules connected to a microcontroller (c).

Figure 6. Hardware set-up for sending the GSM signal from the outside to the inside of an echoic chamber.

Global System for Mobile Phone, GSM; real time clock, RTC; Inter Int grated Circuit, I²C.

Blood sera collection

Venous blood is drawn from the tail of each subject which has been mechanically constrained without anesthesia. The tail is heated for vasolidation and better venous visbiility which could be performed by using heating chamber, heating pad, or heating lamp or by immersing in a warm water. Avoid excessive heating; the animal should be under the lamp no longer than three minutes with a distance of at least 30 cm. All equipment should be sterile, and the skin surface shoul be firstly cleaned and dinsinfected with alcohol 70%. Immobilize the non-dominant tail by hand, and rotate ¼ circle to access the lateral tail vein. The needle is aligned with the tail to 30° for the insertion into the distal vein. Fixation is applied slowly and evenly, and the needle is removed after finished. Observation should be carried out continously during the study period on the subjects’ behavior associated with the ongoing pain.

End point and organ collection

The euthanasia should be carried out by professionals. The rats are physically euthanized by cervical dislocation by applying a firm pressure using the thumb and index finger placed on the posterior skull base and spinal cord. Another hand holds the tail part for a quick backward pulling to dislocate the thoracic vertebrae. Check the respiratory arrest and the absence of heartbeat. Thereafter, the organs are harvested by dissection.

Determinations of serum TSH, T₄, and MDA

The collected blood sample is stored in an EDTA-containing tube at 2–8°C for maximum 5 days. Blood sample should not be hemolyzed or lipemic. The determinations of serum TSH and T4 were performed on ELISA using commercial kit specialized for Rattus novergicus. Normal ranges for serum TSH and T₄ are 0.4–6.0 μIU/mL and 5.0–12.0 μg/dL, respectively. The level of MDA will be measured using ELISA.

Determination of PMCA concentration

Brain tissue, along with the cortex, is collected and rinsed with ice-cold sucrose solution 0.25 M. The tissue is slices and homogenized with 10× operant in a slow setting. The specimen is further treated with Dounce homogenizer as many as 3× operant in a fast speed setting. Dissolution is then carried out to obtain homogenate 6% w/v which is further centrifuged for 10 min at 1.464 × g. The resultant pellet is resuspended in isolating medium and subsequently diluted to yield suspension 6% w/v. As many as 10.4 mL of the suspension is mixed with 1.4 mL Percoll in 15 mL cortex tube and centrifuged for 30 min at 35.540 × g. Two layers are formed on the top side of the type, which are then collected and washed with 5 volumes of sucrose 0.25 M, HEPES-KOH 5 mM, pH 7.1. The obtained pellet is resuspended using the same medium before analyzed using ELISA.

Determination of MCT8 concentration

The whole pituitary gland is collected and frozen in insopentana on dry ice. The sample is melted on slide superfrost (Thermoscientific) and stored at –80°C. The expression is determined based on immunohistochemical method using ELISA.

Determination of brain THRA mRNA expression

Cryosections are performed on the brain tissue, which is then melted on slide superfrost (Thermoscientific) before undergoing a quick freezing on dry ice and being stored at –80°C. Primary antibodies used for the expression determination are mouse antiβ-tubIII, mouse anticalbindin 1:2000, mouse anti –NeuN 1:500, mouse anti-PV 1:2000. Hybridization is carried out in situ at 25 μm part of the juvenile brain. Riboprobes sense and antisense are synthesized from cDNA RC3 template with spanning nucleotide of 253-486. The expression is based on relative expression in the quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis.

Determination of pyruvate kinase concentration

The cerebral cortex is dissected on an ice-cold glass plate and washed in a buffer (sucrose 0.32 M: EGTA 1 mM: Tris-HCL 10 M; pH 7.4). The sample is sliced and homogenized in the aforementioned buffer (1:20 w/v) with Potter-Elvehjem glass homogenizer. The homogenate is centrifuged 10,000 × g for 15 min at 4°C in a Sorval centrifuge. The obtained mitochondria-free supernatant is collected for the enzymatic activity investigation using ELISA.

Determination of NR3C1 selected gene mRNA

As much as 5 mL venous blood is drawn from each sample, treated with EDTA to prevent the coagulation. DNA genome is extracted from the blood using Gentra Puregene Blood Kit following the manufacturer’s manuals. The DNA sample is stored at –20°C until further use. Indirect determination of the gene expression is carried out using RT-qPCR.

Histopathological analysis on thyroid gland

A whole thyroid gland is collected from the subject through dissection, and subsequently fixated using formalin and paraffin for 24 h. Incision is performed on the thyroid gland with a length of 5 μm and the staining is carried out using hematoxylin and eosin following the standardized procedure. Assessment on thyroid follicles is performed under a light microscope. The assessment include:

• Thyroid epithelial cells appeared in a flat shape indicates the hypofunction of TSH.
• Increased form of thyroid epithelial cells indicates the increased function of TSH.
• Transformation of the thyroid epithelial cells into malignant phenotypes.

Expected results and discussion

We expect the reduction of serum TSH and T₄ levels and histological alterations on the thyroid epithelial cells upon the radiation exposure during the nighttime which was performed until 12 weeks. A systematic review on 22 studies reporting the effects of cell phone radiation against thyroid functions suggested the detrimental effects of the exposure via thermal and non-thermal pathways.¹⁹ Exposure of electromagnetic field (900 MHz, 30 min/day for 5 days/week lasted for 4 weeks) was found to significantly decrease serum TSH, T₃, and T₄ (p<0.01), in a double-controlled study.²⁵ Exposure of RF radiation (900 MHz, 1 h/day for 50 days) was reported to significantly increase serum T₄ and decrease T₃ level in Syrian golden hamsters.²⁶ Significant decreases in T₃ and T₄ were observed in chicken embryos and in the newly hatched chicks, but not in older subjects.²³ Enlarged and damaged follicular structures of thyroid was reported in a study exposing a 900 MHz pulse-modulated RF radiation on rats for three weeks (20 min/day).²⁷ No significant effect on thyroid secretory functions, however, is reported from a study investigating sub-chronic whole-body exposure of 915 MHz radiation on rat model.²⁴ In conclusion, T₃ levels were consistently reported to be decreased in previous studies using animal model,²⁵^,²⁶ but not in the case of T₄.²⁶ Furthermore, the effect appeared to be different depending the life stage of the subject (such as embryogenesis).²³ Among, the aforementioned study, only one study considered the cage modification to allow even distribution of the exposure throughout the subject’s body.²⁴

From this present protocol study, the electromagnetic wave radiation exposure is expected to elevate the plasma MDA as a marker of the oxidation stress. This is as indicator of non-thermal effect of electromagnetic wave radiation. In a previous study, radiofrequency emitted from cellular phone has been reported to induce oxidative DNA damage in a rat model, with significant increase in MDA level.²⁸ Ovarian follicles of mice have been reported to experience oxidative stress induced by cell phone radiation with a significantly elevated level of MDA.²⁹ Since brain is sensitive to electromagnetic wave radiation, we expected that the exposure will reduce the TSH concentration followed by T4. The reduction of T4 activities in the brain will be confirmed by reduction of concentrations of MCT8, pyruvate kinase and PMCA as well as reduction of THRA activities. The reduction of NR3C1 activities will be also measured to prove that there is an additional pathway how electromagnetic wave radiation affects the T4 activities (in circulation or in the brain) apart from thermal and non-thermal pathways. It has been suggested that pathologies of thyroid involve Ca²⁺ signaling pathways.³⁰ A scoping review has suggested that Ca²⁺ signaling pathway is disrupted by the cellular phone radiation.³¹ Increased pyruvate kinase activities are associated with thyroid cancer occurrences.³² These along with dysregulation of NR3C1, MCT8 and THRA expressions are indicators for the carcinogenic effects of the cellular phone radiation.³³^,³⁴ Finally, we also expected the histology changes of thyroid gland due to thermal or non-thermal effects of electromagnetic wave radiation or reduction of the TSH concentration that potentially have carcinogenic effects.

Results from the previously reported study might be biased from the exposure of other radiations which has polluted the environment. On contrary, we have observed that the anechoic chamber allows the radiation exposure to exclusively originated from the source used during the investigation (by observing the amplitude and frequency of the radiation using spectrum analyzer). Moreover, the anechoic chamber has a significantly lower electromagnetic field as compared to that in the outside. Thus, the study herein would be performed in anechoic chamber preventing the radiation from the outside to influence the investigation. Moreover, our present study would employ 120—180 min/day exposure for 12 consecutive weeks. This exposure duration is relatively longer than reported in previous studies,²³^,²⁵^,²⁶ though it is lower than that in a study investigating the sub-chronic exposure (8 h/day for, 5 day/week, and lasted for 2—16 weeks).²⁴ Taken altogether, this present study could provide strong evidence pertaining to the effect of mobile phone-derived electromagnetic radiation on the thyroid functions.

Ethics

The animal study protocol was approved by the Institutional Ethics Committee of Faculty of Veterinary Medicine, Universitas Syiah Kuala (Ref No. 69/KEPH/XII/2020).

Study status

The study has not been completed. The exposure of the electromagnetic wave radiation has been conducted and only some of indicators that have been examined.

Author contributions

Conceptualization, H.Z.; methodology, H.Z.; validation, A.R., D.W.S., S.P.S., K.M., H.S. and K.M.; investigation, H.Z.; resources, H.Z.; writing—original draft preparation, H.Z.; writing—review and editing, A.R., D.W.S., S.P.S., K.M., H.S. and K.M.; visualization, H.Z.; supervision, A.R., D.W.S., S.P.S., K.M., H.S. and K.M.; funding acquisition, H.Z. All authors have read and agreed to the published version of the manuscript.

Acknowledgments

We would like to thank all the staff at the Faculty of Veterinary Medicine and all the staff at the Thermal, Vibration, and Acoustic Laboratory, Universitas Syiah Kuala for their assistance during the study.

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Version 2

VERSION 2 PUBLISHED 03 Feb 2023

Author details Author details

Hendra Zufry
Roles: Conceptualization, Funding Acquisition, Investigation, Methodology, Resources, Visualization, Writing – Original Draft Preparation

Achmad Rudijanto
Roles: Supervision, Validation, Writing – Review & Editing

Djoko Wahono Soeatmadji
Roles: Supervision, Validation, Writing – Review & Editing

Setyawan Purnomo Sakti
Roles: Supervision, Validation, Writing – Review & Editing

Khairul Munadi
Roles: Supervision, Validation, Writing – Review & Editing

Hidayat Sujuti
Roles: Supervision, Validation, Writing – Review & Editing

Karyono Mintaroem
Roles: Supervision, Validation, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

The author(s) declared that no grants were involved in supporting this work.

Article Versions (2)

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Published: 09 May 2023, 12:132

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Zufry H, Rudijanto A, Soeatmadji DW et al. A study protocol for investigating the effects of mobile phone-originated electromagnetic waves on thyroid gland and thyroid hormone activities in the brain [version 1; peer review: 2 approved with reservations]. F1000Research 2023, 12:132 (https://doi.org/10.12688/f1000research.129735.1)

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Reviewer Report 06 Apr 2023

I Made Pande Dwipayana, Division of Endocrinology and Metabolism, Department of Internal Medicine, Faculty of Medicine, Sanglah General Hospital, Udayana University, Bali, Indonesia

Approved with Reservations

https://doi.org/10.5256/f1000research.142438.r164252

The introduction of this paper is good and includes the research background. Please add the background of selecting several laboratory parameters examined in this study (i.e. MDA, PMCA, MCT8, etc) and the relationship with thyroid gland an/or

The introduction of this paper is good and includes the research background. Please add the background of selecting several laboratory parameters examined in this study (i.e. MDA, PMCA, MCT8, etc) and the relationship with thyroid gland an/or thyroid hormone activities in the brain. Please provide also clear research question/aim of the study in the introduction.
The design of the study protocol is appropriate. Please add statistical analysis that will be used to prove the hypothesis.
Please provide the reason or the formula for calculating the number of animals in each group in the study to make sure that the number of of sample is adequate to provide the statistical power.
This study has advantages because the use of anechoic chamber since this setting could reduce unwanted electromagnetic waves. The absorption rate is also measured using clear formula.
Multiple indicators are assessed and determined from TSH, T4, MDA, PMCA, MCT8, pyruvate kinase, mRNA expression of THRA and NR3CI genes. The methods being used are appropriate.

Overall the article is good and only need minor revision.

Is the rationale for, and objectives of, the study clearly described?

Yes
Is the study design appropriate for the research question?

Yes
Are sufficient details of the methods provided to allow replication by others?

Yes
Are the datasets clearly presented in a useable and accessible format?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Endocrinology, metabolism, diabetes

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

CITE

Report a concern

Author Response 09 May 2023

Hendra Zufry , Doctoral Program in Medical Sciences, Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia

09 May 2023

Author Response

1. The introduction of this paper is good and includes the research background. Please add the background of selecting several laboratory parameters examined in this study (i.e. MDA, PMCA, MCT8, ... Continue reading 1. The introduction of this paper is good and includes the research background. Please add the background of selecting several laboratory parameters examined in this study (i.e. MDA, PMCA, MCT8, etc) and the relationship with thyroid gland an/or thyroid hormone activities in the brain.

RESPONSES:
Thank you for the suggestion. We have made the following amendment: “Herein, other than T 4, and TSH, the effect of electromagnetic wave radiation will be assessed on several biomolecules including malondialdehyde (MDA), nuclear receptor subfamily 3 group C member 1 (NR3C1), monocarboxylate transporter 8 (MCT8), pyruvate kinase, plasma membrane Ca 2+ ATPase (PMCA), and thyroid hormone receptor alpha (THRA) would be determined after the exposure. MDA acts as the biomarker for oxidative stress induced by the electromagnetic wave radiation. Based on our unpublished preliminary study using computational modelling, NR3C1 is suggested to mediate the oxidative damage on cells and morphometric of thyroid gland, thyroid functions (T 4, and TSH), MCT8, non-genetic thyroid hormones-affected proteins (pyruvate kinase and PMCA) and genetic thyroid hormones-affected protein in the brain (THRA).”

As mentioned above, gene expression of THRA in the brain is affected by the thyroid hormones.

2. Please provide also clear research question/aim of the study in the introduction.

RESPONSES:
Amended, please see below: “The aim is to provide holistic evidence on the effect of electromagnetic wave radiation on thyroid gland and thyroid functions with improved study design that is not bias

ed from the influence of the external radiation by employing anechoic chamber.”

3. The design of the study protocol is appropriate. Please add statistical analysis that will be used to prove the hypothesis.

RESPONSES:
Thank you. The following paragraph has been added:

Statistical analysis
Bivariate analysis will be performed using Spearman test and Chi-square test for continuous and categorical data, respectively. The correlations between the intermediary variables and MDA level or NR3C1 expression will be established using ordinal regression and logistic regression. Statistical significance is considered achieved if p<0.05. All statistical analysis will be performed on IBM SPSS Statistics 23.0 (Armonk, NY, USA).

4. Please provide the reason or the formula for calculating the number of animals in each group in the study to make sure that the number of sample is adequate to provide the statistical power.

RESPONSES:
Thank you. The following amendment has been made:

Sample size
To ensure the statistical power will be achieved, the sample size is determined by Federer’s equation ([n – 1][t – 1] ≥15); where n is the number of sample per group and t is the number of groups. In this study, the number of groups is four, so that six animals are at least required for each group

5. This study has advantages because the use of anechoic chamber since this setting could reduce unwanted electromagnetic waves. The absorption rate is also measured using clear formula.

RESPONSES:
Thank you.

6. Multiple indicators are assessed and determined from TSH, T4, MDA, PMCA, MCT8, pyruvate kinase, mRNA expression of THRA and NR3CI genes. The methods being used are appropriate.

RESPONSES:
Thank you.
1. The introduction of this paper is good and includes the research background. Please add the background of selecting several laboratory parameters examined in this study (i.e. MDA, PMCA, MCT8, etc) and the relationship with thyroid gland an/or thyroid hormone activities in the brain.

RESPONSES:
Thank you for the suggestion. We have made the following amendment: “Herein, other than T 4, and TSH, the effect of electromagnetic wave radiation will be assessed on several biomolecules including malondialdehyde (MDA), nuclear receptor subfamily 3 group C member 1 (NR3C1), monocarboxylate transporter 8 (MCT8), pyruvate kinase, plasma membrane Ca 2+ ATPase (PMCA), and thyroid hormone receptor alpha (THRA) would be determined after the exposure. MDA acts as the biomarker for oxidative stress induced by the electromagnetic wave radiation. Based on our unpublished preliminary study using computational modelling, NR3C1 is suggested to mediate the oxidative damage on cells and morphometric of thyroid gland, thyroid functions (T 4, and TSH), MCT8, non-genetic thyroid hormones-affected proteins (pyruvate kinase and PMCA) and genetic thyroid hormones-affected protein in the brain (THRA).”

As mentioned above, gene expression of THRA in the brain is affected by the thyroid hormones.

2. Please provide also clear research question/aim of the study in the introduction.

RESPONSES:
Amended, please see below: “The aim is to provide holistic evidence on the effect of electromagnetic wave radiation on thyroid gland and thyroid functions with improved study design that is not bias

ed from the influence of the external radiation by employing anechoic chamber.”

3. The design of the study protocol is appropriate. Please add statistical analysis that will be used to prove the hypothesis.

RESPONSES:
Thank you. The following paragraph has been added:

Statistical analysis
Bivariate analysis will be performed using Spearman test and Chi-square test for continuous and categorical data, respectively. The correlations between the intermediary variables and MDA level or NR3C1 expression will be established using ordinal regression and logistic regression. Statistical significance is considered achieved if p<0.05. All statistical analysis will be performed on IBM SPSS Statistics 23.0 (Armonk, NY, USA).

4. Please provide the reason or the formula for calculating the number of animals in each group in the study to make sure that the number of sample is adequate to provide the statistical power.

RESPONSES:
Thank you. The following amendment has been made:

Sample size
To ensure the statistical power will be achieved, the sample size is determined by Federer’s equation ([n – 1][t – 1] ≥15); where n is the number of sample per group and t is the number of groups. In this study, the number of groups is four, so that six animals are at least required for each group

5. This study has advantages because the use of anechoic chamber since this setting could reduce unwanted electromagnetic waves. The absorption rate is also measured using clear formula.

RESPONSES:
Thank you.

6. Multiple indicators are assessed and determined from TSH, T4, MDA, PMCA, MCT8, pyruvate kinase, mRNA expression of THRA and NR3CI genes. The methods being used are appropriate.

RESPONSES:
Thank you.
Competing Interests: None Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 09 May 2023

Hendra Zufry , Doctoral Program in Medical Sciences, Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia

09 May 2023

Author Response

1. The introduction of this paper is good and includes the research background. Please add the background of selecting several laboratory parameters examined in this study (i.e. MDA, PMCA, MCT8, ... Continue reading 1. The introduction of this paper is good and includes the research background. Please add the background of selecting several laboratory parameters examined in this study (i.e. MDA, PMCA, MCT8, etc) and the relationship with thyroid gland an/or thyroid hormone activities in the brain.

RESPONSES:
Thank you for the suggestion. We have made the following amendment: “Herein, other than T 4, and TSH, the effect of electromagnetic wave radiation will be assessed on several biomolecules including malondialdehyde (MDA), nuclear receptor subfamily 3 group C member 1 (NR3C1), monocarboxylate transporter 8 (MCT8), pyruvate kinase, plasma membrane Ca 2+ ATPase (PMCA), and thyroid hormone receptor alpha (THRA) would be determined after the exposure. MDA acts as the biomarker for oxidative stress induced by the electromagnetic wave radiation. Based on our unpublished preliminary study using computational modelling, NR3C1 is suggested to mediate the oxidative damage on cells and morphometric of thyroid gland, thyroid functions (T 4, and TSH), MCT8, non-genetic thyroid hormones-affected proteins (pyruvate kinase and PMCA) and genetic thyroid hormones-affected protein in the brain (THRA).”

As mentioned above, gene expression of THRA in the brain is affected by the thyroid hormones.

2. Please provide also clear research question/aim of the study in the introduction.

RESPONSES:
Amended, please see below: “The aim is to provide holistic evidence on the effect of electromagnetic wave radiation on thyroid gland and thyroid functions with improved study design that is not bias

ed from the influence of the external radiation by employing anechoic chamber.”

3. The design of the study protocol is appropriate. Please add statistical analysis that will be used to prove the hypothesis.

RESPONSES:
Thank you. The following paragraph has been added:

Statistical analysis
Bivariate analysis will be performed using Spearman test and Chi-square test for continuous and categorical data, respectively. The correlations between the intermediary variables and MDA level or NR3C1 expression will be established using ordinal regression and logistic regression. Statistical significance is considered achieved if p<0.05. All statistical analysis will be performed on IBM SPSS Statistics 23.0 (Armonk, NY, USA).

4. Please provide the reason or the formula for calculating the number of animals in each group in the study to make sure that the number of sample is adequate to provide the statistical power.

RESPONSES:
Thank you. The following amendment has been made:

Sample size
To ensure the statistical power will be achieved, the sample size is determined by Federer’s equation ([n – 1][t – 1] ≥15); where n is the number of sample per group and t is the number of groups. In this study, the number of groups is four, so that six animals are at least required for each group

5. This study has advantages because the use of anechoic chamber since this setting could reduce unwanted electromagnetic waves. The absorption rate is also measured using clear formula.

RESPONSES:
Thank you.

6. Multiple indicators are assessed and determined from TSH, T4, MDA, PMCA, MCT8, pyruvate kinase, mRNA expression of THRA and NR3CI genes. The methods being used are appropriate.

RESPONSES:
Thank you.
1. The introduction of this paper is good and includes the research background. Please add the background of selecting several laboratory parameters examined in this study (i.e. MDA, PMCA, MCT8, etc) and the relationship with thyroid gland an/or thyroid hormone activities in the brain.

RESPONSES:
Thank you for the suggestion. We have made the following amendment: “Herein, other than T 4, and TSH, the effect of electromagnetic wave radiation will be assessed on several biomolecules including malondialdehyde (MDA), nuclear receptor subfamily 3 group C member 1 (NR3C1), monocarboxylate transporter 8 (MCT8), pyruvate kinase, plasma membrane Ca 2+ ATPase (PMCA), and thyroid hormone receptor alpha (THRA) would be determined after the exposure. MDA acts as the biomarker for oxidative stress induced by the electromagnetic wave radiation. Based on our unpublished preliminary study using computational modelling, NR3C1 is suggested to mediate the oxidative damage on cells and morphometric of thyroid gland, thyroid functions (T 4, and TSH), MCT8, non-genetic thyroid hormones-affected proteins (pyruvate kinase and PMCA) and genetic thyroid hormones-affected protein in the brain (THRA).”

As mentioned above, gene expression of THRA in the brain is affected by the thyroid hormones.

2. Please provide also clear research question/aim of the study in the introduction.

RESPONSES:
Amended, please see below: “The aim is to provide holistic evidence on the effect of electromagnetic wave radiation on thyroid gland and thyroid functions with improved study design that is not bias

ed from the influence of the external radiation by employing anechoic chamber.”

3. The design of the study protocol is appropriate. Please add statistical analysis that will be used to prove the hypothesis.

RESPONSES:
Thank you. The following paragraph has been added:

Statistical analysis
Bivariate analysis will be performed using Spearman test and Chi-square test for continuous and categorical data, respectively. The correlations between the intermediary variables and MDA level or NR3C1 expression will be established using ordinal regression and logistic regression. Statistical significance is considered achieved if p<0.05. All statistical analysis will be performed on IBM SPSS Statistics 23.0 (Armonk, NY, USA).

4. Please provide the reason or the formula for calculating the number of animals in each group in the study to make sure that the number of sample is adequate to provide the statistical power.

RESPONSES:
Thank you. The following amendment has been made:

Sample size
To ensure the statistical power will be achieved, the sample size is determined by Federer’s equation ([n – 1][t – 1] ≥15); where n is the number of sample per group and t is the number of groups. In this study, the number of groups is four, so that six animals are at least required for each group

5. This study has advantages because the use of anechoic chamber since this setting could reduce unwanted electromagnetic waves. The absorption rate is also measured using clear formula.

RESPONSES:
Thank you.

6. Multiple indicators are assessed and determined from TSH, T4, MDA, PMCA, MCT8, pyruvate kinase, mRNA expression of THRA and NR3CI genes. The methods being used are appropriate.

RESPONSES:
Thank you.
Competing Interests: None Close
Report a concern

Views

Reviewer Report 31 Mar 2023

Alicja Bortkiewicz, Nofer Institute of Occupational Medicine, Łódź, Poland

Piotr Politański, Nofer Institute of Occupational Medicine, Lodz, Poland

Approved with Reservations

https://doi.org/10.5256/f1000research.142438.r164254

The protocol seems well thought out and described, but I have a few doubts and reservations.

In "Introduction" it would also be worth quoting meta-analyses of research on head and neck cancers, and not only two selected original papers Carlberg & Hardell (2012) and Hardell L, Carlberg M, Mild KH. (2013)

My basic doubts concern the planning of the exposure.

Exposure information is inconsistent. In the "Intervension" section, it is stated that the signal frequency will be 1800 MHz, while in the "Source of radio frequency" chapter, the authors state that "The signal of radio frequency (RF) in the anechoic chamber has a frequency of 891—893 MHz". At what distance from the antenna will the cages with animals be placed? This is not clear if all 10 rats from each group are exposed simultaneously. If so, the EMF generated by the antenna will be disturbed. The same problem occurs due to metal mesh on the top of the cage.

Moreover the antenna is not fully omnidirectional and its characteristic is unknown, so EMF in close field, inside the cage should be measured, not approximated. Dimensions of the cage do not guarantee uniform field inside, and 40 dB difference in signal within 10 cm distance (Tab. 1) further reinforces doubts of field uniformity. SAR is quantified for various tissues but with one average (?) density and conductivity and independently of animal relative position to antenna. This is totally unacceptable. Likewise depth of penetration is quantified in equation 2 for throat tissue (what kind of tissue is it) but it is strongly dependent on the skin impedance, which on the other hand is used in equation 3. Data on tissue parameters and used equations should be supported by valid literature. The reasons for chosen times of exposure (2, 2.5 and 3 h) is questionable. The reason for independent presentation of the study protocol itself without any results is unclear and suggests artificial invention of topics, unless this protocol is in the project stage. But in this case reviewer strongly recommends correction of the protocol basing of before mentioned comments.

Minor remarks
Where do the mathematical formulas come from, please indicate the source.
Below table 1, explain the abbreviations MW and dBM

Is the rationale for, and objectives of, the study clearly described?

Yes
Is the study design appropriate for the research question?

No
Are sufficient details of the methods provided to allow replication by others?

No
Are the datasets clearly presented in a useable and accessible format?

Not applicable

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Influence of electromagnetic fields, noise, shiftwork on health especially on cardiovascular system and autonomic nervous system, work-related diseases (mainly cardiovascular diseases), evaluation of the fitness to work in drivers, evaluation of work-fatigue, physical fitness

We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.

CITE

Report a concern

Author Response 09 May 2023

Hendra Zufry , Doctoral Program in Medical Sciences, Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia

09 May 2023

Author Response

Major remarks

1. The protocol seems well thought out and described, but I have a few doubts and reservations.

In "Introduction" it would also be worth quoting meta-analyses ... Continue reading Major remarks

1. The protocol seems well thought out and described, but I have a few doubts and reservations.

In "Introduction" it would also be worth quoting meta-analyses of research on head and neck cancers, and not only two selected original papers Carlberg & Hardell (2012) and Hardell L, Carlberg M, Mild KH. (2013)

RESPONSES:
Thank you. We have tried our best in addressing your queries. Thank you for this well thought suggestion. We have now cited a meta-analysis on the tumor risks associated with electromagnetic radiation.

“A meta-analysis of case-control studies concluded that mobile phone radiation is associated with tumor risks, especially when its usage frequency is > 1000 h.x”

Ref:
Choi Y-J, Moskowitz JM, Myung S-K, Lee YR, Hong Y-C: Cellular Phone use and risk of tumors: Systematic review and meta-analysis. Int. J. Environ. Res. Public Health. 2020;17(21):8079. 33147845 10.3390/ijerph17218079

2. My basic doubts concern the planning of the exposure.

Exposure information is inconsistent. In the "Intervension" section, it is stated that the signal frequency will be 1800 MHz, while in the "Source of radio frequency" chapter, the authors state that "The signal of radio frequency (RF) in the anechoic chamber has a frequency of 891—893 MHz".

RESPONSES:
Thank you for your concern and sorry for the confusion. Let us clarify. The design was pre-planned, and 1800 MHz is the maximum frequency for 3G spectrum. However, when later tested, we found the frequency was ranged 891-893 MHz. Therefore, we have made revision to resolve this confusion:

“The radiation is produced from the stimulation of GSM multiband 4G LTE signal with a frequency of 891—893 MHz (maximum frequency for 3G spectrum is1800 MHz)”

3. At what distance from the antenna will the cages with animals be placed?

RESPONSES:
Antenna will be placed on the center of the cage. With the cage dimensions being 30 x 19 x 14.4 cm^3 (length x width x height), the furthest distance from the animal to the antenna is therefore 19 cm. In the text we have added the following:

“Each cage is divided into five segments with the antenna placed in the center.”

4. This is not clear if all 10 rats from each group are exposed simultaneously. If so, the EMF generated by the antenna will be disturbed. The same problem occurs due to metal mesh on the top of the cage.

RESPONSES:
Thank you for your concern regarding the EMF disturbance. We placed the antenna in the center inside the cage separated into five segments. The small-diameter metal mesh covering the top of the cage was therefore considered to give insignificant effect to the electromagnetic exposure.

And regarding the simultaneous exposure, our apology for the confusion. Please find our revision as follow: “Each cage is divided into five segments with the antenna placed in the center. Each segment will consist of a single rat, and each group will receive simultaneous exposure by employing two separated cages to avoid the disturbance of electromagnetic field.”

4. Moreover, the antenna is not fully omnidirectional and its characteristic is unknown, so EMF in close field, inside the cage should be measured, not approximated. Dimensions of the cage do not guarantee uniform field inside, and 40 dB difference in signal within 10 cm distance (Tab. 1) further reinforces doubts of field uniformity.

RESPONSES:
Thank you for your concern. As seen in Tab 1, we have measured the radiation power from the antenna in the echoic chamber in various distances. The measurement is performed directly by spectrum analyzer, so it is not mathematically estimated.

We believe that the uniformity of the field is achieved since we used real hand-phone concept which allows the omnidirectional property of the antenna – capable of receiving the call from any directions.

Regarding the effect of cage design on the field uniformity, we have already explained it above.

5. SAR is quantified for various tissues but with one average (?) density and conductivity and independently of animal relative position to antenna. This is totally unacceptable. Likewise depth of penetration is quantified in equation 2 for throat tissue (what kind of tissue is it) but it is strongly dependent on the skin impedance, which on the other hand is used in equation 3.

RESPONSES:
It’s true that the value is dependent on the antenna—animal distance. 10-cm distance was taken as the most probable distance of antenna—animal (considering the furthest distance the animal can get from the antenna is 19 cm).

Regarding the SAR value, we understand your concern that the value could be different for each tissue. Our aim is to investigate the effect on thyroid gland; unfortunately, relative permeability and conductivity for thyroid gland is not available. Therefore, we assume the values are equal to that in tissues nearby (namely trachea and lung) which have been known. Clarifications have been made in the following paragraph (highlighted in red).

Where σ is electrical conductivity in the observed tissue (S/m), E is electrical field (V/m), and ρ is the density of body tissue (kg/m 3).

In the real world, the value of ρ is varied, considering each rat has differences in their skin, fat, bones, and so on. Herein, the body density of each rat is assumed to be 1000 kg/m 3. Since the value of σ in thyroid gland is unknown, we assume the value is equal to that in the tissue nearby, namely trachea and lung (σ = 1 S/m for 2 GHz frequency). Since the The value is applicable for rat with the size of around 20 cm × 4.5 cm × 4.5 cm with an averaged body weight of around 200 g.

Where δ is the depth of RF penetration (m), μ is permeability of the observed tissue (H/m), and f is averaged RF signal frequencies (mW). Similar to tissue conductivity, the value of permeability for thyroid gland is currently unknown and therefore is assumed to be equal with that of trachea.

6. Data on tissue parameters and used equations should be supported by valid literature.

RESPONSES:
Thank you. It is true that the calculation protocol should be supported by valid literature. We have now cited it in the paragraph. Please see the amendment: “By inserting all the values required in Equation 1, the SAR is obtained to be 0.0962 W/kg. Equations and biological parameter constants used in calculating the SAR are derived from a previously published report.” Citing:

Shi, J, Chakarothai J, Wang J, Wake K, Watanabe S, Fujiwara O (2014) Quantification and Verification of Whole body average SAR in small animal exposed to electromagnetic field inside reverberation chamber. IEICE Transaction on Cummnunication, E97B(10),2184-2191. https://doi.org/10.1587/transcom.E97.B.2187

7. The reasons for chosen times of exposure (2, 2.5 and 3 h) is questionable.

RESPONSES:
It is based on our unpublished preliminary study. The clarification has been included:
“The daily exposure duration chosen in this research is based on the results from our preliminary, where changes thyroid functions and other metabolic parameters occurred from 120 to 150 min/day exposure (unpublished). In this study, 180 min/day exposure duration will be employed to observe new findings.”

8. The reason for independent presentation of the study protocol itself without any results is unclear and suggests artificial invention of topics, unless this protocol is in the project stage. But in this case reviewer strongly recommends correction of the protocol basing of before mentioned comments.

RESPONSES:
With due respect, we do not intend to make the publication as ‘artificial invention of topics’. Our reasons: Publishing this research as a ‘protocol article’ allows its detailed presentation. Further, this is an on-going project where some data have been collected in the preliminary stage. We thank reviewer for their inputs allowing us to perform research with better methodology.

Minor remarks
1. Where do the mathematical formulas come from, please indicate the source.

RESPONSES:
References used:
1.         Chakarothai J, Wang J, Fujiwara O, Wake K & Watanabe S (2013).Dosimetri of reverberation chamber for whole-body exposure of small animal.IEEE Transactions on microwave Theory and Techniques, 61(9),3435-3445.
2.         Shi, J, Chakarothai J, Wang J, Wake K, Watanabe S, Fujiwara O (2014) Quantification and Verificationof Whole body average SAR in small animal exposed to electromagnetic field inside reverberation chamber. IEICE Transaction on Cummnunication, E97B(10),2184-2191. https://doi.org/10.1587/transcom.E97.B.2187

These references have been cited in the text as in the previous response.

2. Below table 1, explain the abbreviations MW and dBM.

RESPONSES:
Revised.
In the Table 1 footnote:
mW ; miliwatt, dBm : The power ratio in decibels (dB) of the measured power referenced to one milliwatt (mW).
Major remarks

1. The protocol seems well thought out and described, but I have a few doubts and reservations.

In "Introduction" it would also be worth quoting meta-analyses of research on head and neck cancers, and not only two selected original papers Carlberg & Hardell (2012) and Hardell L, Carlberg M, Mild KH. (2013)

RESPONSES:
Thank you. We have tried our best in addressing your queries. Thank you for this well thought suggestion. We have now cited a meta-analysis on the tumor risks associated with electromagnetic radiation.

“A meta-analysis of case-control studies concluded that mobile phone radiation is associated with tumor risks, especially when its usage frequency is > 1000 h.x”

Ref:
Choi Y-J, Moskowitz JM, Myung S-K, Lee YR, Hong Y-C: Cellular Phone use and risk of tumors: Systematic review and meta-analysis. Int. J. Environ. Res. Public Health. 2020;17(21):8079. 33147845 10.3390/ijerph17218079

2. My basic doubts concern the planning of the exposure.

Exposure information is inconsistent. In the "Intervension" section, it is stated that the signal frequency will be 1800 MHz, while in the "Source of radio frequency" chapter, the authors state that "The signal of radio frequency (RF) in the anechoic chamber has a frequency of 891—893 MHz".

RESPONSES:
Thank you for your concern and sorry for the confusion. Let us clarify. The design was pre-planned, and 1800 MHz is the maximum frequency for 3G spectrum. However, when later tested, we found the frequency was ranged 891-893 MHz. Therefore, we have made revision to resolve this confusion:

“The radiation is produced from the stimulation of GSM multiband 4G LTE signal with a frequency of 891—893 MHz (maximum frequency for 3G spectrum is1800 MHz)”

3. At what distance from the antenna will the cages with animals be placed?

RESPONSES:
Antenna will be placed on the center of the cage. With the cage dimensions being 30 x 19 x 14.4 cm^3 (length x width x height), the furthest distance from the animal to the antenna is therefore 19 cm. In the text we have added the following:

“Each cage is divided into five segments with the antenna placed in the center.”

4. This is not clear if all 10 rats from each group are exposed simultaneously. If so, the EMF generated by the antenna will be disturbed. The same problem occurs due to metal mesh on the top of the cage.

RESPONSES:
Thank you for your concern regarding the EMF disturbance. We placed the antenna in the center inside the cage separated into five segments. The small-diameter metal mesh covering the top of the cage was therefore considered to give insignificant effect to the electromagnetic exposure.

And regarding the simultaneous exposure, our apology for the confusion. Please find our revision as follow: “Each cage is divided into five segments with the antenna placed in the center. Each segment will consist of a single rat, and each group will receive simultaneous exposure by employing two separated cages to avoid the disturbance of electromagnetic field.”

4. Moreover, the antenna is not fully omnidirectional and its characteristic is unknown, so EMF in close field, inside the cage should be measured, not approximated. Dimensions of the cage do not guarantee uniform field inside, and 40 dB difference in signal within 10 cm distance (Tab. 1) further reinforces doubts of field uniformity.

RESPONSES:
Thank you for your concern. As seen in Tab 1, we have measured the radiation power from the antenna in the echoic chamber in various distances. The measurement is performed directly by spectrum analyzer, so it is not mathematically estimated.

We believe that the uniformity of the field is achieved since we used real hand-phone concept which allows the omnidirectional property of the antenna – capable of receiving the call from any directions.

Regarding the effect of cage design on the field uniformity, we have already explained it above.

5. SAR is quantified for various tissues but with one average (?) density and conductivity and independently of animal relative position to antenna. This is totally unacceptable. Likewise depth of penetration is quantified in equation 2 for throat tissue (what kind of tissue is it) but it is strongly dependent on the skin impedance, which on the other hand is used in equation 3.

RESPONSES:
It’s true that the value is dependent on the antenna—animal distance. 10-cm distance was taken as the most probable distance of antenna—animal (considering the furthest distance the animal can get from the antenna is 19 cm).

Regarding the SAR value, we understand your concern that the value could be different for each tissue. Our aim is to investigate the effect on thyroid gland; unfortunately, relative permeability and conductivity for thyroid gland is not available. Therefore, we assume the values are equal to that in tissues nearby (namely trachea and lung) which have been known. Clarifications have been made in the following paragraph (highlighted in red).

Where σ is electrical conductivity in the observed tissue (S/m), E is electrical field (V/m), and ρ is the density of body tissue (kg/m 3).

In the real world, the value of ρ is varied, considering each rat has differences in their skin, fat, bones, and so on. Herein, the body density of each rat is assumed to be 1000 kg/m 3. Since the value of σ in thyroid gland is unknown, we assume the value is equal to that in the tissue nearby, namely trachea and lung (σ = 1 S/m for 2 GHz frequency). Since the The value is applicable for rat with the size of around 20 cm × 4.5 cm × 4.5 cm with an averaged body weight of around 200 g.

Where δ is the depth of RF penetration (m), μ is permeability of the observed tissue (H/m), and f is averaged RF signal frequencies (mW). Similar to tissue conductivity, the value of permeability for thyroid gland is currently unknown and therefore is assumed to be equal with that of trachea.

6. Data on tissue parameters and used equations should be supported by valid literature.

RESPONSES:
Thank you. It is true that the calculation protocol should be supported by valid literature. We have now cited it in the paragraph. Please see the amendment: “By inserting all the values required in Equation 1, the SAR is obtained to be 0.0962 W/kg. Equations and biological parameter constants used in calculating the SAR are derived from a previously published report.” Citing:

Shi, J, Chakarothai J, Wang J, Wake K, Watanabe S, Fujiwara O (2014) Quantification and Verification of Whole body average SAR in small animal exposed to electromagnetic field inside reverberation chamber. IEICE Transaction on Cummnunication, E97B(10),2184-2191. https://doi.org/10.1587/transcom.E97.B.2187

7. The reasons for chosen times of exposure (2, 2.5 and 3 h) is questionable.

RESPONSES:
It is based on our unpublished preliminary study. The clarification has been included:
“The daily exposure duration chosen in this research is based on the results from our preliminary, where changes thyroid functions and other metabolic parameters occurred from 120 to 150 min/day exposure (unpublished). In this study, 180 min/day exposure duration will be employed to observe new findings.”

8. The reason for independent presentation of the study protocol itself without any results is unclear and suggests artificial invention of topics, unless this protocol is in the project stage. But in this case reviewer strongly recommends correction of the protocol basing of before mentioned comments.

RESPONSES:
With due respect, we do not intend to make the publication as ‘artificial invention of topics’. Our reasons: Publishing this research as a ‘protocol article’ allows its detailed presentation. Further, this is an on-going project where some data have been collected in the preliminary stage. We thank reviewer for their inputs allowing us to perform research with better methodology.

Minor remarks
1. Where do the mathematical formulas come from, please indicate the source.

RESPONSES:
References used:
1.         Chakarothai J, Wang J, Fujiwara O, Wake K & Watanabe S (2013).Dosimetri of reverberation chamber for whole-body exposure of small animal.IEEE Transactions on microwave Theory and Techniques, 61(9),3435-3445.
2.         Shi, J, Chakarothai J, Wang J, Wake K, Watanabe S, Fujiwara O (2014) Quantification and Verificationof Whole body average SAR in small animal exposed to electromagnetic field inside reverberation chamber. IEICE Transaction on Cummnunication, E97B(10),2184-2191. https://doi.org/10.1587/transcom.E97.B.2187

These references have been cited in the text as in the previous response.

2. Below table 1, explain the abbreviations MW and dBM.

RESPONSES:
Revised.
In the Table 1 footnote:
mW ; miliwatt, dBm : The power ratio in decibels (dB) of the measured power referenced to one milliwatt (mW).
Competing Interests: None Close
Report a concern
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COMMENTS ON THIS REPORT

Author Response 09 May 2023

Hendra Zufry , Doctoral Program in Medical Sciences, Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia

09 May 2023

Author Response

Major remarks

1. The protocol seems well thought out and described, but I have a few doubts and reservations.

In "Introduction" it would also be worth quoting meta-analyses ... Continue reading Major remarks

1. The protocol seems well thought out and described, but I have a few doubts and reservations.

In "Introduction" it would also be worth quoting meta-analyses of research on head and neck cancers, and not only two selected original papers Carlberg & Hardell (2012) and Hardell L, Carlberg M, Mild KH. (2013)

RESPONSES:
Thank you. We have tried our best in addressing your queries. Thank you for this well thought suggestion. We have now cited a meta-analysis on the tumor risks associated with electromagnetic radiation.

“A meta-analysis of case-control studies concluded that mobile phone radiation is associated with tumor risks, especially when its usage frequency is > 1000 h.x”

Ref:
Choi Y-J, Moskowitz JM, Myung S-K, Lee YR, Hong Y-C: Cellular Phone use and risk of tumors: Systematic review and meta-analysis. Int. J. Environ. Res. Public Health. 2020;17(21):8079. 33147845 10.3390/ijerph17218079

2. My basic doubts concern the planning of the exposure.

Exposure information is inconsistent. In the "Intervension" section, it is stated that the signal frequency will be 1800 MHz, while in the "Source of radio frequency" chapter, the authors state that "The signal of radio frequency (RF) in the anechoic chamber has a frequency of 891—893 MHz".

RESPONSES:
Thank you for your concern and sorry for the confusion. Let us clarify. The design was pre-planned, and 1800 MHz is the maximum frequency for 3G spectrum. However, when later tested, we found the frequency was ranged 891-893 MHz. Therefore, we have made revision to resolve this confusion:

“The radiation is produced from the stimulation of GSM multiband 4G LTE signal with a frequency of 891—893 MHz (maximum frequency for 3G spectrum is1800 MHz)”

3. At what distance from the antenna will the cages with animals be placed?

RESPONSES:
Antenna will be placed on the center of the cage. With the cage dimensions being 30 x 19 x 14.4 cm^3 (length x width x height), the furthest distance from the animal to the antenna is therefore 19 cm. In the text we have added the following:

“Each cage is divided into five segments with the antenna placed in the center.”

4. This is not clear if all 10 rats from each group are exposed simultaneously. If so, the EMF generated by the antenna will be disturbed. The same problem occurs due to metal mesh on the top of the cage.

RESPONSES:
Thank you for your concern regarding the EMF disturbance. We placed the antenna in the center inside the cage separated into five segments. The small-diameter metal mesh covering the top of the cage was therefore considered to give insignificant effect to the electromagnetic exposure.

And regarding the simultaneous exposure, our apology for the confusion. Please find our revision as follow: “Each cage is divided into five segments with the antenna placed in the center. Each segment will consist of a single rat, and each group will receive simultaneous exposure by employing two separated cages to avoid the disturbance of electromagnetic field.”

4. Moreover, the antenna is not fully omnidirectional and its characteristic is unknown, so EMF in close field, inside the cage should be measured, not approximated. Dimensions of the cage do not guarantee uniform field inside, and 40 dB difference in signal within 10 cm distance (Tab. 1) further reinforces doubts of field uniformity.

RESPONSES:
Thank you for your concern. As seen in Tab 1, we have measured the radiation power from the antenna in the echoic chamber in various distances. The measurement is performed directly by spectrum analyzer, so it is not mathematically estimated.

We believe that the uniformity of the field is achieved since we used real hand-phone concept which allows the omnidirectional property of the antenna – capable of receiving the call from any directions.

Regarding the effect of cage design on the field uniformity, we have already explained it above.

5. SAR is quantified for various tissues but with one average (?) density and conductivity and independently of animal relative position to antenna. This is totally unacceptable. Likewise depth of penetration is quantified in equation 2 for throat tissue (what kind of tissue is it) but it is strongly dependent on the skin impedance, which on the other hand is used in equation 3.

RESPONSES:
It’s true that the value is dependent on the antenna—animal distance. 10-cm distance was taken as the most probable distance of antenna—animal (considering the furthest distance the animal can get from the antenna is 19 cm).

Regarding the SAR value, we understand your concern that the value could be different for each tissue. Our aim is to investigate the effect on thyroid gland; unfortunately, relative permeability and conductivity for thyroid gland is not available. Therefore, we assume the values are equal to that in tissues nearby (namely trachea and lung) which have been known. Clarifications have been made in the following paragraph (highlighted in red).

Where σ is electrical conductivity in the observed tissue (S/m), E is electrical field (V/m), and ρ is the density of body tissue (kg/m 3).

In the real world, the value of ρ is varied, considering each rat has differences in their skin, fat, bones, and so on. Herein, the body density of each rat is assumed to be 1000 kg/m 3. Since the value of σ in thyroid gland is unknown, we assume the value is equal to that in the tissue nearby, namely trachea and lung (σ = 1 S/m for 2 GHz frequency). Since the The value is applicable for rat with the size of around 20 cm × 4.5 cm × 4.5 cm with an averaged body weight of around 200 g.

Where δ is the depth of RF penetration (m), μ is permeability of the observed tissue (H/m), and f is averaged RF signal frequencies (mW). Similar to tissue conductivity, the value of permeability for thyroid gland is currently unknown and therefore is assumed to be equal with that of trachea.

6. Data on tissue parameters and used equations should be supported by valid literature.

RESPONSES:
Thank you. It is true that the calculation protocol should be supported by valid literature. We have now cited it in the paragraph. Please see the amendment: “By inserting all the values required in Equation 1, the SAR is obtained to be 0.0962 W/kg. Equations and biological parameter constants used in calculating the SAR are derived from a previously published report.” Citing:

Shi, J, Chakarothai J, Wang J, Wake K, Watanabe S, Fujiwara O (2014) Quantification and Verification of Whole body average SAR in small animal exposed to electromagnetic field inside reverberation chamber. IEICE Transaction on Cummnunication, E97B(10),2184-2191. https://doi.org/10.1587/transcom.E97.B.2187

7. The reasons for chosen times of exposure (2, 2.5 and 3 h) is questionable.

RESPONSES:
It is based on our unpublished preliminary study. The clarification has been included:
“The daily exposure duration chosen in this research is based on the results from our preliminary, where changes thyroid functions and other metabolic parameters occurred from 120 to 150 min/day exposure (unpublished). In this study, 180 min/day exposure duration will be employed to observe new findings.”

8. The reason for independent presentation of the study protocol itself without any results is unclear and suggests artificial invention of topics, unless this protocol is in the project stage. But in this case reviewer strongly recommends correction of the protocol basing of before mentioned comments.

RESPONSES:
With due respect, we do not intend to make the publication as ‘artificial invention of topics’. Our reasons: Publishing this research as a ‘protocol article’ allows its detailed presentation. Further, this is an on-going project where some data have been collected in the preliminary stage. We thank reviewer for their inputs allowing us to perform research with better methodology.

Minor remarks
1. Where do the mathematical formulas come from, please indicate the source.

RESPONSES:
References used:
1.         Chakarothai J, Wang J, Fujiwara O, Wake K & Watanabe S (2013).Dosimetri of reverberation chamber for whole-body exposure of small animal.IEEE Transactions on microwave Theory and Techniques, 61(9),3435-3445.
2.         Shi, J, Chakarothai J, Wang J, Wake K, Watanabe S, Fujiwara O (2014) Quantification and Verificationof Whole body average SAR in small animal exposed to electromagnetic field inside reverberation chamber. IEICE Transaction on Cummnunication, E97B(10),2184-2191. https://doi.org/10.1587/transcom.E97.B.2187

These references have been cited in the text as in the previous response.

2. Below table 1, explain the abbreviations MW and dBM.

RESPONSES:
Revised.
In the Table 1 footnote:
mW ; miliwatt, dBm : The power ratio in decibels (dB) of the measured power referenced to one milliwatt (mW).
Major remarks

1. The protocol seems well thought out and described, but I have a few doubts and reservations.

In "Introduction" it would also be worth quoting meta-analyses of research on head and neck cancers, and not only two selected original papers Carlberg & Hardell (2012) and Hardell L, Carlberg M, Mild KH. (2013)

RESPONSES:
Thank you. We have tried our best in addressing your queries. Thank you for this well thought suggestion. We have now cited a meta-analysis on the tumor risks associated with electromagnetic radiation.

“A meta-analysis of case-control studies concluded that mobile phone radiation is associated with tumor risks, especially when its usage frequency is > 1000 h.x”

Ref:
Choi Y-J, Moskowitz JM, Myung S-K, Lee YR, Hong Y-C: Cellular Phone use and risk of tumors: Systematic review and meta-analysis. Int. J. Environ. Res. Public Health. 2020;17(21):8079. 33147845 10.3390/ijerph17218079

2. My basic doubts concern the planning of the exposure.

Exposure information is inconsistent. In the "Intervension" section, it is stated that the signal frequency will be 1800 MHz, while in the "Source of radio frequency" chapter, the authors state that "The signal of radio frequency (RF) in the anechoic chamber has a frequency of 891—893 MHz".

RESPONSES:
Thank you for your concern and sorry for the confusion. Let us clarify. The design was pre-planned, and 1800 MHz is the maximum frequency for 3G spectrum. However, when later tested, we found the frequency was ranged 891-893 MHz. Therefore, we have made revision to resolve this confusion:

“The radiation is produced from the stimulation of GSM multiband 4G LTE signal with a frequency of 891—893 MHz (maximum frequency for 3G spectrum is1800 MHz)”

3. At what distance from the antenna will the cages with animals be placed?

RESPONSES:
Antenna will be placed on the center of the cage. With the cage dimensions being 30 x 19 x 14.4 cm^3 (length x width x height), the furthest distance from the animal to the antenna is therefore 19 cm. In the text we have added the following:

“Each cage is divided into five segments with the antenna placed in the center.”

4. This is not clear if all 10 rats from each group are exposed simultaneously. If so, the EMF generated by the antenna will be disturbed. The same problem occurs due to metal mesh on the top of the cage.

RESPONSES:
Thank you for your concern regarding the EMF disturbance. We placed the antenna in the center inside the cage separated into five segments. The small-diameter metal mesh covering the top of the cage was therefore considered to give insignificant effect to the electromagnetic exposure.

And regarding the simultaneous exposure, our apology for the confusion. Please find our revision as follow: “Each cage is divided into five segments with the antenna placed in the center. Each segment will consist of a single rat, and each group will receive simultaneous exposure by employing two separated cages to avoid the disturbance of electromagnetic field.”

4. Moreover, the antenna is not fully omnidirectional and its characteristic is unknown, so EMF in close field, inside the cage should be measured, not approximated. Dimensions of the cage do not guarantee uniform field inside, and 40 dB difference in signal within 10 cm distance (Tab. 1) further reinforces doubts of field uniformity.

RESPONSES:
Thank you for your concern. As seen in Tab 1, we have measured the radiation power from the antenna in the echoic chamber in various distances. The measurement is performed directly by spectrum analyzer, so it is not mathematically estimated.

We believe that the uniformity of the field is achieved since we used real hand-phone concept which allows the omnidirectional property of the antenna – capable of receiving the call from any directions.

Regarding the effect of cage design on the field uniformity, we have already explained it above.

5. SAR is quantified for various tissues but with one average (?) density and conductivity and independently of animal relative position to antenna. This is totally unacceptable. Likewise depth of penetration is quantified in equation 2 for throat tissue (what kind of tissue is it) but it is strongly dependent on the skin impedance, which on the other hand is used in equation 3.

RESPONSES:
It’s true that the value is dependent on the antenna—animal distance. 10-cm distance was taken as the most probable distance of antenna—animal (considering the furthest distance the animal can get from the antenna is 19 cm).

Regarding the SAR value, we understand your concern that the value could be different for each tissue. Our aim is to investigate the effect on thyroid gland; unfortunately, relative permeability and conductivity for thyroid gland is not available. Therefore, we assume the values are equal to that in tissues nearby (namely trachea and lung) which have been known. Clarifications have been made in the following paragraph (highlighted in red).

Where σ is electrical conductivity in the observed tissue (S/m), E is electrical field (V/m), and ρ is the density of body tissue (kg/m 3).

In the real world, the value of ρ is varied, considering each rat has differences in their skin, fat, bones, and so on. Herein, the body density of each rat is assumed to be 1000 kg/m 3. Since the value of σ in thyroid gland is unknown, we assume the value is equal to that in the tissue nearby, namely trachea and lung (σ = 1 S/m for 2 GHz frequency). Since the The value is applicable for rat with the size of around 20 cm × 4.5 cm × 4.5 cm with an averaged body weight of around 200 g.

Where δ is the depth of RF penetration (m), μ is permeability of the observed tissue (H/m), and f is averaged RF signal frequencies (mW). Similar to tissue conductivity, the value of permeability for thyroid gland is currently unknown and therefore is assumed to be equal with that of trachea.

6. Data on tissue parameters and used equations should be supported by valid literature.

RESPONSES:
Thank you. It is true that the calculation protocol should be supported by valid literature. We have now cited it in the paragraph. Please see the amendment: “By inserting all the values required in Equation 1, the SAR is obtained to be 0.0962 W/kg. Equations and biological parameter constants used in calculating the SAR are derived from a previously published report.” Citing:

Shi, J, Chakarothai J, Wang J, Wake K, Watanabe S, Fujiwara O (2014) Quantification and Verification of Whole body average SAR in small animal exposed to electromagnetic field inside reverberation chamber. IEICE Transaction on Cummnunication, E97B(10),2184-2191. https://doi.org/10.1587/transcom.E97.B.2187

7. The reasons for chosen times of exposure (2, 2.5 and 3 h) is questionable.

RESPONSES:
It is based on our unpublished preliminary study. The clarification has been included:
“The daily exposure duration chosen in this research is based on the results from our preliminary, where changes thyroid functions and other metabolic parameters occurred from 120 to 150 min/day exposure (unpublished). In this study, 180 min/day exposure duration will be employed to observe new findings.”

8. The reason for independent presentation of the study protocol itself without any results is unclear and suggests artificial invention of topics, unless this protocol is in the project stage. But in this case reviewer strongly recommends correction of the protocol basing of before mentioned comments.

RESPONSES:
With due respect, we do not intend to make the publication as ‘artificial invention of topics’. Our reasons: Publishing this research as a ‘protocol article’ allows its detailed presentation. Further, this is an on-going project where some data have been collected in the preliminary stage. We thank reviewer for their inputs allowing us to perform research with better methodology.

Minor remarks
1. Where do the mathematical formulas come from, please indicate the source.

RESPONSES:
References used:
1.         Chakarothai J, Wang J, Fujiwara O, Wake K & Watanabe S (2013).Dosimetri of reverberation chamber for whole-body exposure of small animal.IEEE Transactions on microwave Theory and Techniques, 61(9),3435-3445.
2.         Shi, J, Chakarothai J, Wang J, Wake K, Watanabe S, Fujiwara O (2014) Quantification and Verificationof Whole body average SAR in small animal exposed to electromagnetic field inside reverberation chamber. IEICE Transaction on Cummnunication, E97B(10),2184-2191. https://doi.org/10.1587/transcom.E97.B.2187

These references have been cited in the text as in the previous response.

2. Below table 1, explain the abbreviations MW and dBM.

RESPONSES:
Revised.
In the Table 1 footnote:
mW ; miliwatt, dBm : The power ratio in decibels (dB) of the measured power referenced to one milliwatt (mW).
Competing Interests: None Close
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Version 2

VERSION 2 PUBLISHED 03 Feb 2023

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Version 2 (revision) 09 May 23	read	read
Version 1 03 Feb 23	read	read

Alicja Bortkiewicz, Nofer Institute of Occupational Medicine, Łódź, Poland

Piotr Politański, Nofer Institute of Occupational Medicine, Lodz, Poland
I Made Pande Dwipayana, Udayana University, Bali, Indonesia

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25 May 2023 | for Version 2

Alicja Bortkiewicz, Nofer Institute of Occupational Medicine, Łódź, Poland

Piotr Politański, Nofer Institute of Occupational Medicine, Lodz, Poland

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Not Approved

Responses provided by Authors do not satisfy reviewers. They rather increase the concern about the misunderstanding of the topic by the Authors. Papers cited as an explanation of chosen equations do not contain chosen equations. Those papers quantify with Method of Moments rectangular dielectric phantom whole body SAR for some specific exposure conditions. In this case conditions are totally unknown. Coarse measurements with mysterious “spectrum analyzer” with undescribed antenna of unknown properties is not the way of obtaining valid exposure data. Assumption, that metal mesh on the top on the cage do not disturb EMF inside the cage is as far from reality as possible. Moreover Authors provided proofs, that all exposure system provides not uniform EMF field. Results from table 1 show about 40 dB difference between measurements made in 20 and 30 cm from antenna, wherein higher value was measured further from the antenna. This all proves that Authors do not understand EMF basics.

Authors wrote ”Different and non-standardized protocols used in investigating the aforementioned effects have made scientist difficult to reach a conclusive finding. Of which, previous studies were unable to exclude the effects of non-ionizing electromagnetic radiation from outside the system. Indeed, a cage modification has been proposed in a study using anechoic chamber allowing whole-body exposure of the electromagnetic field.²⁵ Nonetheless, the possible bias from the external electromagnetic wave influence persists. Therefore, we reported the use of anechoic chamber as a mean to prevent the influence of the external radiation during the investigation.”– using anechoic chamber is not the way of building perfect exposure system by itself. There is necessity of providing uniform EMF in which tested animals would be placed and therefore exposed inside of this chamber, and this part was omitted by authors.

Furthermore all chapter devoted to SAR quantifications is full of mistakes, discrepancies or misspellings:

“To calculate SARS (SAR’s) value, it is of importance to calculate the depth of RF signal penetration using the following equation […] Where δ is the depth of RF penetration (m), μ is permeability of the observed tissue (H/m), and f is averaged RF signal frequencies (mW). Similar to tissue conductivity, the value of permeability for thyroid gland is currently unknown and therefore is assumed to be equal with that of trachea. In the case of RF penetration into throat tissue, the δ (σ?) is 1.1 and the averaged uplink frequency for GSM is 9902.5 MHz(990.25?). Therefore, by using the Equation 2 we obtain the value for δ = 0.0159 m. – in further calculations this very important signal penetration depth was not used at all – so what was the importance?

In a GSM 900 system the maximum power is 2 W; hence, the maximum averaged power (P) is 250 mW (1/8 maximum power). this is close to the truth only in case of the GSM and in real life situations its rather ¼ maximum power, as in GSM system power is transmitted in two of the eight time slots (technical slot and voice transmission slot), but Authors stated before that The signal of radio frequency (RF) in the anechoic chamber has a frequency of 891—893 MHz(discrepancy with previous paragraph where 990,25 MHz (?) was mentioned) (GSM and UMTS signals) with Gaussian Minimum-Shift Keying (GMSK) modulation scheme for GSM, Quadrature Phase Shift Keying (QPSK) – UMTS, and 8PSK – EDGE, so one can assume as the UMTS signal is not transmitted in time slots, that maximum power would be 2 W.

If the averaged distance between the phone antenna and rat (A) is 10 cm, the radiation density with maximal penetration (I) could be calculated using Equation 3 (I=P/A)resulting in I = 1.481 W/m².– how Watts divided by meters could possibly result in Watts per square meter is another inexplicable conundrum. Electrical current(none of presented equations does not apply to electric current! – this is equation for Power density) is expressed by Equation 4 which can be derived to Equation 5 by substituting P. With the skin impedance (η) value of 59.094 Ohm, the electrical field strength (E) could be calculated using Equation 5 resulting in E = 9.355 V/m. By inserting all the values required in Equation 1, the SAR is obtained to be 0.0962 W/kg.

Estimated SAR will vary not only due to difference in external field, but also due to the orientation of the animal. Also whole body SAR will vary from SAR averaged in some particular tissue. And reviewers may only wish that ways of quantification would be as simple as provided by authors, but this is only a wishful thinking. Those are EMF basics, and without them no paper should be considered for publishing. In the first review Reviewers strongly suggested correction of the protocol before indexing and this suggestion must be sustained. In its present form the paper must not be approved as the exposure system do not guarantee uniform (or at least known) exposure and SAR’s quantifications are mistaken at best.

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No competing interests were disclosed.

We confirm that we have read this submission and believe that we have an appropriate level of expertise to state that we do not consider it to be of an acceptable scientific standard, for reasons outlined above.

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15 May 2023 | for Version 2

I Made Pande Dwipayana, Division of Endocrinology and Metabolism, Department of Internal Medicine, Faculty of Medicine, Sanglah General Hospital, Udayana University, Bali, Indonesia

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Approved

The authors have revised the article and have addressed all of the concerns or feedback that I have given.

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No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Reviewer Report

10 Views

06 Apr 2023 | for Version 1

I Made Pande Dwipayana, Division of Endocrinology and Metabolism, Department of Internal Medicine, Faculty of Medicine, Sanglah General Hospital, Udayana University, Bali, Indonesia

10 Views Cite this report Responses(1)

Approved With Reservations

The introduction of this paper is good and includes the research background. Please add the background of selecting several laboratory parameters examined in this study (i.e. MDA, PMCA, MCT8, etc) and the relationship with thyroid gland an/or thyroid hormone activities in the brain. Please provide also clear research question/aim of the study in the introduction.
The design of the study protocol is appropriate. Please add statistical analysis that will be used to prove the hypothesis.
Please provide the reason or the formula for calculating the number of animals in each group in the study to make sure that the number of of sample is adequate to provide the statistical power.
This study has advantages because the use of anechoic chamber since this setting could reduce unwanted electromagnetic waves. The absorption rate is also measured using clear formula.
Multiple indicators are assessed and determined from TSH, T4, MDA, PMCA, MCT8, pyruvate kinase, mRNA expression of THRA and NR3CI genes. The methods being used are appropriate.

Overall the article is good and only need minor revision.

Is the rationale for, and objectives of, the study clearly described?

Yes
Is the study design appropriate for the research question?

Yes
Are sufficient details of the methods provided to allow replication by others?

Yes
Are the datasets clearly presented in a useable and accessible format?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Endocrinology, metabolism, diabetes

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Author Response

09 May 2023

Hendra Zufry , Doctoral Program in Medical Sciences, Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia

1. The introduction of this paper is good and includes the research background. Please add the background of selecting several laboratory parameters examined in this study (i.e. MDA, PMCA, MCT8, etc) and the relationship with thyroid gland an/or thyroid hormone activities in the brain.

RESPONSES:
Thank you for the suggestion. We have made the following amendment: “Herein, other than T 4, and TSH, the effect of electromagnetic wave radiation will be assessed on several biomolecules including malondialdehyde (MDA), nuclear receptor subfamily 3 group C member 1 (NR3C1), monocarboxylate transporter 8 (MCT8), pyruvate kinase, plasma membrane Ca 2+ ATPase (PMCA), and thyroid hormone receptor alpha (THRA) would be determined after the exposure. MDA acts as the biomarker for oxidative stress induced by the electromagnetic wave radiation. Based on our unpublished preliminary study using computational modelling, NR3C1 is suggested to mediate the oxidative damage on cells and morphometric of thyroid gland, thyroid functions (T 4, and TSH), MCT8, non-genetic thyroid hormones-affected proteins (pyruvate kinase and PMCA) and genetic thyroid hormones-affected protein in the brain (THRA).”

As mentioned above, gene expression of THRA in the brain is affected by the thyroid hormones.

2. Please provide also clear research question/aim of the study in the introduction.

RESPONSES:
Amended, please see below: “The aim is to provide holistic evidence on the effect of electromagnetic wave radiation on thyroid gland and thyroid functions with improved study design that is not bias

ed from the influence of the external radiation by employing anechoic chamber.”

3. The design of the study protocol is appropriate. Please add statistical analysis that will be used to prove the hypothesis.

RESPONSES:
Thank you. The following paragraph has been added:

Statistical analysis
Bivariate analysis will be performed using Spearman test and Chi-square test for continuous and categorical data, respectively. The correlations between the intermediary variables and MDA level or NR3C1 expression will be established using ordinal regression and logistic regression. Statistical significance is considered achieved if p<0.05. All statistical analysis will be performed on IBM SPSS Statistics 23.0 (Armonk, NY, USA).

4. Please provide the reason or the formula for calculating the number of animals in each group in the study to make sure that the number of sample is adequate to provide the statistical power.

RESPONSES:
Thank you. The following amendment has been made:

Sample size
To ensure the statistical power will be achieved, the sample size is determined by Federer’s equation ([n – 1][t – 1] ≥15); where n is the number of sample per group and t is the number of groups. In this study, the number of groups is four, so that six animals are at least required for each group

5. This study has advantages because the use of anechoic chamber since this setting could reduce unwanted electromagnetic waves. The absorption rate is also measured using clear formula.

RESPONSES:
Thank you.

6. Multiple indicators are assessed and determined from TSH, T4, MDA, PMCA, MCT8, pyruvate kinase, mRNA expression of THRA and NR3CI genes. The methods being used are appropriate.

RESPONSES:
Thank you.

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Competing Interests

None

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Reviewer Report

17 Views

31 Mar 2023 | for Version 1

Alicja Bortkiewicz, Nofer Institute of Occupational Medicine, Łódź, Poland

Piotr Politański, Nofer Institute of Occupational Medicine, Lodz, Poland

17 Views Cite this report Responses(1)

Approved With Reservations

Is the rationale for, and objectives of, the study clearly described?

Yes
Is the study design appropriate for the research question?

No
Are sufficient details of the methods provided to allow replication by others?

No
Are the datasets clearly presented in a useable and accessible format?

Not applicable

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Influence of electromagnetic fields, noise, shiftwork on health especially on cardiovascular system and autonomic nervous system, work-related diseases (mainly cardiovascular diseases), evaluation of the fitness to work in drivers, evaluation of work-fatigue, physical fitness

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Responses (1)

Author Response

09 May 2023

Hendra Zufry , Doctoral Program in Medical Sciences, Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia

Major remarks

1. The protocol seems well thought out and described, but I have a few doubts and reservations.

In "Introduction" it would also be worth quoting meta-analyses of research on head and neck cancers, and not only two selected original papers Carlberg & Hardell (2012) and Hardell L, Carlberg M, Mild KH. (2013)

RESPONSES:
Thank you. We have tried our best in addressing your queries. Thank you for this well thought suggestion. We have now cited a meta-analysis on the tumor risks associated with electromagnetic radiation.

“A meta-analysis of case-control studies concluded that mobile phone radiation is associated with tumor risks, especially when its usage frequency is > 1000 h.x”

Ref:
Choi Y-J, Moskowitz JM, Myung S-K, Lee YR, Hong Y-C: Cellular Phone use and risk of tumors: Systematic review and meta-analysis. Int. J. Environ. Res. Public Health. 2020;17(21):8079. 33147845 10.3390/ijerph17218079

2. My basic doubts concern the planning of the exposure.

Exposure information is inconsistent. In the "Intervension" section, it is stated that the signal frequency will be 1800 MHz, while in the "Source of radio frequency" chapter, the authors state that "The signal of radio frequency (RF) in the anechoic chamber has a frequency of 891—893 MHz".

RESPONSES:
Thank you for your concern and sorry for the confusion. Let us clarify. The design was pre-planned, and 1800 MHz is the maximum frequency for 3G spectrum. However, when later tested, we found the frequency was ranged 891-893 MHz. Therefore, we have made revision to resolve this confusion:

“The radiation is produced from the stimulation of GSM multiband 4G LTE signal with a frequency of 891—893 MHz (maximum frequency for 3G spectrum is1800 MHz)”

3. At what distance from the antenna will the cages with animals be placed?

RESPONSES:
Antenna will be placed on the center of the cage. With the cage dimensions being 30 x 19 x 14.4 cm^3 (length x width x height), the furthest distance from the animal to the antenna is therefore 19 cm. In the text we have added the following:

“Each cage is divided into five segments with the antenna placed in the center.”

4. This is not clear if all 10 rats from each group are exposed simultaneously. If so, the EMF generated by the antenna will be disturbed. The same problem occurs due to metal mesh on the top of the cage.

RESPONSES:
Thank you for your concern regarding the EMF disturbance. We placed the antenna in the center inside the cage separated into five segments. The small-diameter metal mesh covering the top of the cage was therefore considered to give insignificant effect to the electromagnetic exposure.

And regarding the simultaneous exposure, our apology for the confusion. Please find our revision as follow: “Each cage is divided into five segments with the antenna placed in the center. Each segment will consist of a single rat, and each group will receive simultaneous exposure by employing two separated cages to avoid the disturbance of electromagnetic field.”

4. Moreover, the antenna is not fully omnidirectional and its characteristic is unknown, so EMF in close field, inside the cage should be measured, not approximated. Dimensions of the cage do not guarantee uniform field inside, and 40 dB difference in signal within 10 cm distance (Tab. 1) further reinforces doubts of field uniformity.

RESPONSES:
Thank you for your concern. As seen in Tab 1, we have measured the radiation power from the antenna in the echoic chamber in various distances. The measurement is performed directly by spectrum analyzer, so it is not mathematically estimated.

We believe that the uniformity of the field is achieved since we used real hand-phone concept which allows the omnidirectional property of the antenna – capable of receiving the call from any directions.

Regarding the effect of cage design on the field uniformity, we have already explained it above.

5. SAR is quantified for various tissues but with one average (?) density and conductivity and independently of animal relative position to antenna. This is totally unacceptable. Likewise depth of penetration is quantified in equation 2 for throat tissue (what kind of tissue is it) but it is strongly dependent on the skin impedance, which on the other hand is used in equation 3.

RESPONSES:
It’s true that the value is dependent on the antenna—animal distance. 10-cm distance was taken as the most probable distance of antenna—animal (considering the furthest distance the animal can get from the antenna is 19 cm).

Regarding the SAR value, we understand your concern that the value could be different for each tissue. Our aim is to investigate the effect on thyroid gland; unfortunately, relative permeability and conductivity for thyroid gland is not available. Therefore, we assume the values are equal to that in tissues nearby (namely trachea and lung) which have been known. Clarifications have been made in the following paragraph (highlighted in red).

Where σ is electrical conductivity in the observed tissue (S/m), E is electrical field (V/m), and ρ is the density of body tissue (kg/m 3).

In the real world, the value of ρ is varied, considering each rat has differences in their skin, fat, bones, and so on. Herein, the body density of each rat is assumed to be 1000 kg/m 3. Since the value of σ in thyroid gland is unknown, we assume the value is equal to that in the tissue nearby, namely trachea and lung (σ = 1 S/m for 2 GHz frequency). Since the The value is applicable for rat with the size of around 20 cm × 4.5 cm × 4.5 cm with an averaged body weight of around 200 g.

Where δ is the depth of RF penetration (m), μ is permeability of the observed tissue (H/m), and f is averaged RF signal frequencies (mW). Similar to tissue conductivity, the value of permeability for thyroid gland is currently unknown and therefore is assumed to be equal with that of trachea.

6. Data on tissue parameters and used equations should be supported by valid literature.

RESPONSES:
Thank you. It is true that the calculation protocol should be supported by valid literature. We have now cited it in the paragraph. Please see the amendment: “By inserting all the values required in Equation 1, the SAR is obtained to be 0.0962 W/kg. Equations and biological parameter constants used in calculating the SAR are derived from a previously published report.” Citing:

Shi, J, Chakarothai J, Wang J, Wake K, Watanabe S, Fujiwara O (2014) Quantification and Verification of Whole body average SAR in small animal exposed to electromagnetic field inside reverberation chamber. IEICE Transaction on Cummnunication, E97B(10),2184-2191. https://doi.org/10.1587/transcom.E97.B.2187

7. The reasons for chosen times of exposure (2, 2.5 and 3 h) is questionable.

RESPONSES:
It is based on our unpublished preliminary study. The clarification has been included:
“The daily exposure duration chosen in this research is based on the results from our preliminary, where changes thyroid functions and other metabolic parameters occurred from 120 to 150 min/day exposure (unpublished). In this study, 180 min/day exposure duration will be employed to observe new findings.”

8. The reason for independent presentation of the study protocol itself without any results is unclear and suggests artificial invention of topics, unless this protocol is in the project stage. But in this case reviewer strongly recommends correction of the protocol basing of before mentioned comments.

RESPONSES:
With due respect, we do not intend to make the publication as ‘artificial invention of topics’. Our reasons: Publishing this research as a ‘protocol article’ allows its detailed presentation. Further, this is an on-going project where some data have been collected in the preliminary stage. We thank reviewer for their inputs allowing us to perform research with better methodology.

Minor remarks
1. Where do the mathematical formulas come from, please indicate the source.

RESPONSES:
References used:
1. Chakarothai J, Wang J, Fujiwara O, Wake K & Watanabe S (2013).Dosimetri of reverberation chamber for whole-body exposure of small animal.IEEE Transactions on microwave Theory and Techniques, 61(9),3435-3445.
2. Shi, J, Chakarothai J, Wang J, Wake K, Watanabe S, Fujiwara O (2014) Quantification and Verificationof Whole body average SAR in small animal exposed to electromagnetic field inside reverberation chamber. IEICE Transaction on Cummnunication, E97B(10),2184-2191. https://doi.org/10.1587/transcom.E97.B.2187

These references have been cited in the text as in the previous response.

2. Below table 1, explain the abbreviations MW and dBM.

RESPONSES:
Revised.
In the Table 1 footnote:
mW ; miliwatt, dBm : The power ratio in decibels (dB) of the measured power referenced to one milliwatt (mW).

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Competing Interests

None

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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A study protocol for investigating the effects of mobile phone-originated electromagnetic waves on thyroid gland and thyroid hormone activities in the brain

Abstract

Keywords

Introduction

Experimental design

Figure 1. Study workflow for investigating the effects of mobile phone-originated electromagnetic wave radiation on thyroid functions.

Equipment

Animal

Anechoic chamber design

Location and chamber design

Figure 2. Photograph of the anechoic chamber in Universitas Syiah Kuala, Indonesia.

Environmental conditions

Signal strength

Figure 3. Equipment set-up for signal strength measurement.

Source of radio frequency

Figure 4. Design (left) and photographed images (right) of the GSM signal sender (a) and receiver (b).

Determination of noise and radiation frequency

Table 1. Results of the measurements of signal frequencies from RF/GSM inside the anechoic chamber.

Design and installation of RF applicator

Determination of specific absorption rate

(1)

(2)

(3)

(4)

(5)

Procedure

Randomization and acclimation

Table 2. Group assignment.

Adaptation

Intervention

Figure 6. Hardware set-up for sending the GSM signal from the outside to the inside of an echoic chamber.

Blood sera collection

End point and organ collection

Determinations of serum TSH, T4, and MDA

Determination of PMCA concentration

Determination of MCT8 concentration

Determination of brain THRA mRNA expression

Determination of pyruvate kinase concentration

Determination of NR3C1 selected gene mRNA

Histopathological analysis on thyroid gland

Expected results and discussion

Ethics

Study status

Author contributions

Acknowledgments

References

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Determinations of serum TSH, T₄, and MDA