Kidney and liver histology in tumour-induced rats exposed to non-contact electric fields

Firman Alamsyah; Nisrina Firdausi; Subekti Evi Dwi Nugraheni; Ahmad Ghitha Fadhlurrahman; Luthfi Nurhidayat; Rarastoeti Pratiwi; Warsito Purwo Taruno

doi:10.12688/f1000research.110080.1

Home Browse Kidney and liver histology in tumour-induced rats exposed to non-contact...

ALL Metrics

Views

Downloads

Get PDF

Get XML

Export

▬

✚

Research Article

Kidney and liver histology in tumour-induced rats exposed to non-contact electric fields

[version 1; peer review: 1 approved, 1 not approved]

Firman Alamsyah ^1,2, Nisrina Firdausi³, Subekti Evi Dwi Nugraheni⁴, [...] Ahmad Ghitha Fadhlurrahman⁵, Luthfi Nurhidayat⁵, Rarastoeti Pratiwi⁵, Warsito Purwo Taruno¹

Firman Alamsyah ^1,2, Nisrina Firdausi³, [...] Subekti Evi Dwi Nugraheni⁴, Ahmad Ghitha Fadhlurrahman⁵, Luthfi Nurhidayat⁵, Rarastoeti Pratiwi⁵, Warsito Purwo Taruno¹

PUBLISHED 01 Feb 2023

Author details Author details

¹ Center for Medical Physics and Cancer Research, Ctech Labs Edwar Technology, Tangerang, Banten, 15143, Indonesia
² Faculty of Science and Technology, Universitas Al-Azhar Indonesia, Jl. Sisingamangaraja, Jakarta, 12110, Indonesia
³ Research Center for Pharmaceutical Ingridients and Traditional Medicine, National Research and Innovation Agency Republic of Indonesia, Jl. Raya Jakarta-Bogor Km. 46, Cibinong West Java, 16911, Indonesia
⁴ Medical Laboratory, Universitas Pendidikan Indonesia, Jl. Dr. Setiabudhi No. 229, Bandung West Java, 40154, Indonesia
⁵ Faculty of Biology, Universitas Gadjah Mada, Sleman, DI Yogyakarta, 55281, Indonesia

Firman Alamsyah
Roles: Conceptualization, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Nisrina Firdausi
Roles: Data Curation, Formal Analysis, Investigation, Methodology, Resources, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Subekti Evi Dwi Nugraheni
Roles: Data Curation, Formal Analysis, Investigation, Methodology, Resources, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Ahmad Ghitha Fadhlurrahman
Roles: Investigation, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing

Luthfi Nurhidayat
Roles: Methodology, Resources, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

Rarastoeti Pratiwi
Roles: Conceptualization, Investigation, Methodology, Resources, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

Warsito Purwo Taruno
Roles: Funding Acquisition, Resources, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Oncology gateway.

Abstract

Background: There were an estimated 2.1 million breast cancer diagnoses in 2018 worldwide, which is about 11.6% of the total cancer incidence. A novel modality of cancer treatment based on exposure to non-contact electric fields has been developed to reduce cancer incidence. However, the safety of the electric field exposure was not fully investigated. Therefore, the purpose of this study is to observe the safety of the electric field exposure on renal and liver structure.
Methods: Female Sprague-Dawley rats were divided into one control group and three treatment groups. Animals were treated with 7,12-dimethylbenz[a]anthracene for mammary tumour induction and exposed to non-contact electric fields individually for 10 hours a day for three weeks. Fresh samples of the kidney and liver were collected for observing structural damage in both organs. The two organs were prepared for histopathological cross-sectioning using the paraffin method and Hematoxylin & Eosin staining followed by histological scoring using the post-examination masking method.
Results: The damages found in the kidney were the following: thickening of Bowman capsule, karyolysis, karyorrexhis, pyknosis, cloudy swelling, epithelial sloughing, inflammation, haemorrhage, and congestion. The number of inflammation and haemorrhage in the kidney structure of the placebo group was the lowest and significantly different from the three other groups. All damages in the kidney were also found in the liver, but each showed different levels of damage. The damages in the kidney and liver caused by the exposure were not significant.
Conclusions: The non-contact electric fields were not harmful to renal and liver structure in tumour-induced rats. Instead, it may increase the renal function in normal rats.

Keywords

damages, histology, kidney, liver, non-contact electric field, ECCT

Corresponding author: Firman Alamsyah

Competing interests: All authors had financial support as a research grant from Ctech Labs Edwar Technology to complete the submitted work; FA and WPT received salary from Ctech Labs Edwar Technology; NF, SEDN and AGF received honorarium from Ctech Labs Edwar Technology; no financial relationships with any organisations that might have interests in the submitted work; no other relationships or activities that could appear to have influenced the submitted work.

Grant information: This study was funded by Ctech Labs Edwar Technology.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2023 Alamsyah F 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: Alamsyah F, Firdausi N, Nugraheni SED et al. Kidney and liver histology in tumour-induced rats exposed to non-contact electric fields [version 1; peer review: 1 approved, 1 not approved]. F1000Research 2023, 12:117 (https://doi.org/10.12688/f1000research.110080.1) First published: 01 Feb 2023, 12:117 (https://doi.org/10.12688/f1000research.110080.1) Latest published: 11 Feb 2025, 12:117 (https://doi.org/10.12688/f1000research.110080.6)

Introduction

The knowledge that electric fields can induce biological effects was revealed in the 19^th century. Many studies were conducted which provided evidence that exposure to electric fields can generate alterations within living things.¹ Some studies have examined the effects generated by electric fields on cell functions.² Kirson et al.³ also reported that electric field intensity within a cell is less than 10 V/cm, but in a cell membrane, it may gain 10⁵ V/cm. At the organ level, the kidney and liver have the dielectric property that exhibits a time-temperature dependence.⁴^–⁶ Therefore, they possess electrical conductivity and permittivity.⁵^,⁶

Porter et al.⁷ explained that the knowledge of dielectric properties of biological tissues is valuable and useful in several applications of medical device, including cancer detection and treatment. As proof of that, the proliferation of cancer cells was successfully inhibited under exposure to intermediate frequency and low-intensity electric fields.³^,⁸^–¹¹ They use intermediate frequency to treat cancer because it specifically targets cancer cells and does not affect normal cells due to their higher membrane potential than that of cancer cells.¹²^,¹³ In our preliminary study, mammary tumour-induced mice that were exposed to intermediate frequency (100 kHz) and low-intensity (18 Volt peak to peak/Vpp) non-contact electric fields showed no histological alterations in mammary and skin tissues.⁸ Furthermore, we developed non-contact electric fields to avoid dermatitis due to the direct contact between the electrodes and the skin, as reported by Kirson et al.³ This novel modality has the potential to decrease the global cancer burden; 2.1 million people around the world were diagnosed with breast cancer in 2018, which is 11.6% of the total cancer incidence.¹⁴

Although non-contact electric fields-based therapy has the potential to treat cancer, the safety of this kind of therapy when treating healthy tissues should be investigated. This is because injuries may occur after exposure to electric fields due to the dielectric property of the kidney and liver, which may interact with electrostatic waves. Therefore, it is important to investigate the abnormalities in the kidney and liver under exposure to electric fields during cancer treatment. The aim of this work was to investigate the safety of non-contact electric fields of strength 100 kHz-18 Vpp on the kidney and liver in the animal tumour model, with a focus on possible histological alterations in the organs. We hypothesised that exposure to non-contact electric fields would not affect the structure of the kidney and liver significantly. According to our knowledge, this is the first study investigating the abnormalities in the kidney and liver under exposure to 100 kHz intermediate frequency and low-intensity non-contact electric fields.

Methods

Experimental design

The experimental design and procedures, experimental animals, animal care and monitoring, housing and husbandry, sample size, inclusion and exclusion criteria, randomisation and blinding in this study were the same as the ones that have been previously reported.⁹ For this study, 40 5-week-old healthy female Sprague Dawley (SD) rats (Rattus norvegicus, Berkenhout 1769) weighing 50−80 g were used. This rat strain is one of the animals used as animal tumor models to study human breast cancer, since it has 98% genetic homology with humans.¹⁵ These rats were provided by the Integrated Research and Testing Laboratory (LPPT) of Universitas Gadjah Mada (UGM), and never used for other studies. Rats that were sick or showing symptoms of disorder were excluded from the study. The rats were placed into polypropylene cages for one week of acclimatization. The cages were communal home cages with a size 50 × 40 cm² and the base was covered with rice hulls bedding. We prepared eight communal cages with each cage consisted of 5 animals. The lighting conditions in the animal’s room during the day came from light from the lamp, while at night it was total darkness (12L:12D photoperiod). We maintained room temperature to avoid dehydration during exposure to the electric field at 23–26°C with an average relative humidity of 81.09%.

We divided the animals into one control group (non-induction and non-therapy or NINT) and three treatment groups, namely placebo (non-induction and therapy or NIT), DMBA-induced mammary tumours without therapy (induction and non-therapy or INT), and DMBA induced mammary tumours with therapy (induction and therapy or IT) group. Using the Federer formula, the sample size in each group was calculated, in which 6 biological replicates were used for each group¹¹ and they were randomly selected to be assigned to the control and treatment groups.⁹

We administered a single dose of 7,12-dimethylbenz[a]anthracene (DMBA), 20 mg/kg body weight, to induce mammary tumours in rats in INT and IT groups. The administration of DMBA was conducted twice weekly for five weeks. This carcinogenic agent has been widely used in many mammary tumour studies using SD rats.¹⁶^,¹⁷ Furthermore, the rats in the NIT and IT groups were treated with exposure to intermediate frequency (100 kHz) and low-intensity (18 Vpp) electric fields for 10 hours daily for 21 days in modified individual cages.⁹ Mammary tumours were palpated every two days with a digital caliper and their size (cm²) was tabulated. Nodule size were not measured in volume due to tool limitations. All tumour measurements were performed by the same investigator (NF). The therapy was terminated once the mammary tumours increased to 2.25 cm² in size or therapy was completed on day 21. All rats were returned to their communal cages every day after therapy had completed.

Individual cages were cleaned daily by removing rat droppings and changing feed and water.⁹ Rat fur was given picric acid as an individual marker to avoid potential confounders, while rat cages were labeled with paint markers as group markers. Each work in this study, such as DMBA administration, euthanised rat dissection, kidney and liver sample fixation and data analysis, was carried out by different investigators. One investigator (FA) controlled and monitored all works in this study.

Necropsy and organ harvesting

After completion of the treatment, all animals were euthanised under anaesthesia using an overdose of ketamine (150 mg/kg of body weight) via intramuscular injection. The animals were dissected ventrally side up on a dissected box by the same surgeon (AGF).⁹ Two kidneys and two liver organs from different rats were collected randomly from each group. These 16 organs were used for histological examination. The number of samples used for histopathological examination was representative.

Renal histopathological analysis

Samples of the left kidney were taken from all groups by necropsy, washed using physiological saline (0.9% NaCl) and then fixed using 10% neutral buffered formalin (NBF). This organ was prepared for histopathological cross-sections using the paraffin method and hematoxylin and eosin (H&E) staining with a slightly modified protocol adapted from Bancroft and Cook.¹⁸ A piece of the organ that has been fixed then dehydrated using ethanol with a grade of 70%, 80%, 90%, and 100% for 2-3 repetitions, then followed by 4 hours clearing process with xylol at room temperature. Furthermore, the organ was infiltrated by putting it in the liquid paraffin at 60^oC for 50 minutes with 3 repetitions. The next step was embedding which is putting the organ in a paraffin mold that contain liquid paraffin, then cooling it at room temperature. Then paraffin block which contains the organ was sectioned with 4-5 μm thickness. Then the organ slices were placed on a glass slide and deparaffinized by dipping them in xylol for 3x5 minutes. Then dehydration was performed using graded alcohol 96%, 90%, 80%, 70%, 50%, and distilled water for 1 minute each. The slides were then dipped in a solution of hematoxylin dye for 2-5 minutes, and dehydrated with 50% and 70% alcohol. Furthermore, it was dipped in eosin dye for 5-10 minutes, and dehydrated with 70%, 80%, 90%, and 96% alcohol. The last step was clearing for 15 minutes in xylol, and finally covered the slide using a cover glass.

Histopathological scoring of the kidneys was performed using the post-examination masking method combined with the ordinal scoring method.¹⁹ The scoring referred to the endothelial-glomerular-tubular-interstitial (EGTI) system²⁰ that adjusted to the research requirements by replacing the endothelial parameter with the number of congestions (Table 1). The scoring was performed on the renal cortex and medulla in 100 fields per group with 40x objective lens magnification. Microphotographs were taken using Leica DM750 photomicrographic microscope. Kidney sample fixation and histopathological analysis were performed by the same researcher (NF).

Table 1. Histopathological scoring system for the kidney.

Tissue type	Injury	Score
Glomerular	No damage	0
	Thickening of Bowman capsule	1
	Retraction of glomerular tuft	2
	Glomerular fibrosis	3
Tubular	No damage	0
	Reversible damage	1
	Reversible damage with necrosis in tissue less than 25%	2
	Reversible damage with necrosis in tissue between 25% and 50%	3
	Reversible damage with necrosis in tissue more than 50%	4
Interstitial	No damage	0
	Inflammation or haemorrhage exists	1
	Inflammation or haemorrhage exists with necrosis in tissue less than 25%	2
	Inflammation or haemorrhage exists with necrosis in tissue between 25% and 60%	3
	Inflammation or haemorrhage exists with necrosis in tissue more than 60%	4
Congestion	No congestion	0
	Congestion in tissue less than 25%	1
	Congestion in tissue between 25% and 50%	2
	Congestion in tissue between 51% and 75%	3
	Congestion in tissue between 76% and 100%	4

Liver histopathological analysis

The liver was washed in physiological saline (0.9% NaCl) and immersed in a fixative solution (10% NBF). The histological preparation of the liver was carried out using the paraffin method of haematoxylin and eosin staining following Bancroft and Cook¹⁸ with the same steps as for kidney preparation. Histopathological scoring was performed using the ordinal post-examination masking method. Scoring was carried out in 100 fields per group using 40x objective lens magnification. Three parameters of damage, namely cellular damage, haemorrhage, and congestion were determined for the histopathological scoring system²¹^–²³ (Table 2). Liver sample fixation and histopathological analysis were performed by the same researcher (SEDN).

Table 2. Histopathological scoring system for the liver.

Tissue type	Injury	Score
Cellular damage	No damage	0
	Reversible damage with necrosis in tissue less than 15%	1
	Reversible damage with necrosis in tissue between 15% and 40%	2
	Reversible damage with necrosis in tissues between 41% and 70%	3
	Reversible damage with necrosis in tissue between 71% and 100%	4
Haemorrhagic	No damage	0
	<15%	1
	15–40%	2
	41–70%	3
	71–100%	4
Congestion	No congestion	0
	Congestion in tissue less than 15%	1
	Congestion in tissue between 15% and 40%	2
	Congestion in tissue between 41% and 70%	3
	Congestion in tissue between 71% and 100%	4

Data analysis

All measured data were analysed using the appropriate methods and without any exclusion. Data were analysed qualitatively and quantitatively. Qualitative data analysis was carried out descriptively. For quantitative data analysis, normality test was carried out first using the Shapiro-Wilk test (α=0.05). The scoring results were then analysed statistically using the Kruskal-Wallis test, which was followed by the Mann-Whitney test (α=0.05), since the data were not normally distributed, to find significant differences among the group (p<0.05). All data were analysed statistically using the SPSS version 16 (RRID:SCR_002865) program by the same researcher (NF).

Results

The outcome of this research was the comparison of the histological characteristics of the kidney and liver under exposure to non-contact electric fields, which will be explained coherently in the sections below.

Histopathology of kidney

The effects of exposure to non-contact electric fields on renal histopathology and the ordinal scoring results of kidney damages are illustrated in Figure 1 and Figure 2, respectively. The main damage found in the kidney glomerular was the thickening of the Bowman capsule whose scores were significant in all treatment groups (1.12±0.56 for NIT, 1.16±0.74 for INT, and 1.24±0.59 for IT groups) compared to the control (NINT) group (0.88±0.56). In the kidney tubules, more damages were found, including karyolysis, karyorrexhis, pyknosis, cloudy swelling, and epithelial sloughing. However, the scores of these injuries were not significantly different among groups. In the kidney interstitial tissues, inflammation and haemorrhage were identified and the score of both damages in the placebo (NIT) group was the lowest (1.0±0.55) and significantly different to three other groups (1.19±0.51for NINT, 1.35±0.63 for INT, and 1.31±0.63 for IT groups). Congestion was found as a common injury in all parts of the kidney structure, and the number of congestions in the kidney structure of the placebo (NIT) group was also the lowest among treatment groups, but were not significantly different from the three other groups.

Figure 1. Histological features of tubular, interstitial, glomerular, and congestion damages in rat renal sections with H&E staining.

KL=Karyolysis, KR=karyorrexhis, PK=pyknosis, CS=cloudy swelling, ES=epithelial sloughing, Co=congestion, In=inflammation, Hm=haemorrhage, TBC=thickening of Bowman’s capsule, NINT=non-induction and non-therapy group, NIT=non-induction and therapy group, INT=induction and non-therapy group, and IT=induction and therapy group.

Figure 2. Scoring of tubular, interstitial, glomerular, and congestion damages in rat renal sections.

(a) Tubular damages, (b) interstitial damages, (c) glomerular damages, and (d) number of congestions.

Histopathology of liver

The histopathological structure of the liver in the four groups had the same damage pattern but with different levels of damage as shown in Figure 3 and Figure 4. All groups had the same type of damage, namely cellular damage (pyknosis, karyolysis, karyorrhexis), haemorrhage and congestion, as well as reversible damage (cellular swelling and fatty change). No significant cellular damages were found after exposure to non-contact electric fields. Instead, the score of cellular injuries and hemorrhage was the highest after DMBA administration in INT group (1.96±0.51 and 0.88±0.46, respectively) and significantly different to control (NINT) group (1.75±0.43 and 0.63±0.48, respectively). The significant difference of hemorrhage score between IT group (0.87±0.56) and control (NINT) group (0.63±0.48),due to DMBA administration. Exposure to electric field in IT group slightly decreased hemorrhage, cellular injuries and congestion in liver (0.87±0.56, 1.82±0.48, 0.37±0.56, respectively) after DMBA administration as compared to INT group (0.88±0.46, 1.96±0.51, 0.52±0.66, respectively). The scores of congestion were also not significantly different among groups. The histology of the liver tissue in all groups did not show fibrosis, so it can be said that the congestion that occurred was not at a chronic level. Since there was no significant difference in the score of congestion among groups and fibrosis were not found, the congestion in all groups was still considered normal.

Figure 3. Histological features of haemorrhage, congestion, and cellular damages in rat liver section with H&E staining.

Hr=Haemorrhage, Cg=congestion, Pn=pyknosis, Kr=karyorrhexis, Kl=karyolysis, Cs=cell swelling, Fc=fatty change, NINT=non-induction and non-therapy group, NIT=non-induction and therapy group, INT=induction and non-therapy group, and IT=induction and therapy group.

Figure 4. Scoring of cellular damages, haemorrhage, and congestion in rat liver sections.

(a) Cellular damages, (b) haemorrhage, and (c) number of congestions.

Discussion

In the present study, the safety of the non-contact electric fields was revealed in the results of the histopathological analysis of kidney and liver in mammary tumour-induced rats, as discussed below.

The thickening of Bowman’s capsule as the main damage in the glomerulus (Figure 1) may be a result of glomerular hyperfiltration,²⁴ nephrotoxicity caused by DMBA administration,²⁵ and exposure to electric fields.²⁶ Since the significant damages of the glomerulus were observed in the kidneys of the placebo (NIT), non-therapy (INT) and therapy (IT) groups, both DMBA administration and non-contact electric field exposure affected the thickening of Bowman’s capsule. Sharma and Paliwal²⁷ reported that kidneys are one of the major target organs of DMBA (nephrotoxicity) and that the epithelial cells of Bowman’s capsule and the proximal convoluted tubules appear to be more susceptible to DMBA. Whereas the electric fields affected this damage by changing the transmembrane potential and distribution of ion channels and dipoles following changes in the membrane structure.²⁸ Although both DMBA administration and exposure to non-contact electric fields affected the thickening of Bowman’s capsule, the individual toxicity effect of the electric field exposure was lower than the one with DMBA administration, as shown in Figure 2. Additionally, some biological effects of exposure to electric fields (0.6 and 340 kV/m) were revealed in humans and vertebrates, but no histological abnormalities were found in the organs, including the kidneys.²⁹ Therefore, DMBA administration would have a greater risk of inducing renal impairment.

The nephrotoxicity effect of DMBA did not occur only in the glomerulus, but also in the tubules. Moreover, DMBA caused substantive nephrotoxicity that is depicted by renal tubular necrosis including karyolysis, karyorrexhis and pyknosis,³⁰ as shown in Figure 1. Additionally, DMBA created obvious reversible histological changes in the tubules, such as epithelial sloughing and cloudy swelling, as illustrated in Figure 1. The epithelial sloughing represented the progressive disintegration of the tubules,²⁹ and the cloudy swelling may lead to cell necrosis.⁴ However, since the score of each injury in the renal tubules was not significantly different among the groups, the nephrotoxicity effect of DMBA and the exposure to non-contact electric fields were not harmful to renal tubules. No reports have revealed necrosis or reversible injuries in renal tubules under exposure to the intermediate frequency and low-intensity electric fields, except exposure to 100 electric pulses resulting in high-intensity 575±67 V/cm electric fields for irreversible electroporation.³¹ Therefore, the exposure to intermediate frequency and low intensity non-contact electric fields was not harmful to the renal tubules.

In renal interstitial tissues, the nephrotoxicity effect of DMBA significantly caused inflammation and haemorrhage, as shown in Figure 2. This inflammation can be affected by oxidative stress and may induce renal function impairment, including endothelial dysfunction, atherosclerosis, and glomerular injury.³² Oxidative stress activates transcription factors including NF-kB, which activate the inflammatory response gene expression.³³ Moreover, Kandeel et al.³⁴ reported that oxidative stress may change the renal structure and function due to the effect of reactive oxygen species (ROS) on mesangial and endothelial cells. Oxidative injury happens when ROS, including O₂, H₂O₂ and -OH, ruin the antioxidant defence systems of the cells.³⁵ This ROS may be produced due to DMBA administration³⁶ and it can spread from the site of production to other sites inside the cells or even extend the injury outside the cells.³⁷ Additionally, de Oliveira et al.³⁸ revealed that DMBA administration to develop a tumour in an animal model also causes haemorrhage. No reports have revealed inflammation and haemorrhage as well as congestion under exposure to intermediate frequency and low-intensity electric fields. Therefore, the exposure to non-contact electric fields was also not harmful to renal interstitial tissues. In fact, non-contact electric fields decreased the number of inflammations and haemorrhages in the placebo (NIT) group, as shown in Figure 2.

Almost the same as in kidney histology, there was no significant damage to the liver after exposure to electric fields (Figure 4). The results in the non-therapy (INT) group with the highest significant rate of hepatocellular damage and hemorrhage indicated that DMBA as a carcinogenic substance can increase the presence of intercellular haemorrhage in the hepatic tissue.²³ Duarte et al.²³ reported mild hepatotoxicity in the liver, including the presence of a pyknotic phase of nuclei of hepatocytes due to DMBA induction. However, haemorrhage in the hepatic tissue has not yet shown symptoms of acute haemorrhage, including cellular hypoxia, decreased tissue perfusion, organ damage, and death.³⁹ The results in the therapy (IT) group with a lower rate of hepatocellular damage compared to the non-therapy (INT) one suggested that exposure to non-contact electric fields had lesser damaging effects than DMBA administration. Additionally, since the value of the congestion of blood vessels was still within the normal condition and not at the chronic level, the exposure to non-contact electric fields was not harmful. It was found that the intensity, frequency, and duration of exposure to non-contact electric fields and the dose of DMBA administered causes changes in the different parameters evaluated, although not very significant ones. However, these changes could be considered as a sign of metabolic alterations under the effect of the exposure to the non-contact electric fields and DMBA administration.

Based on the evidence for the efficacy and safety of normal tissues and organs,⁸^,⁹ including kidney and liver as reported in this study, we will be conducting a phase I clinical trial of ECCT for healthy volunteers using a 100 kHz 18 Vpp electric field as used in this study. Moreover, since this electric fields exposure may decrease the number of inflammations and haemorrhage in kidney, this therapy may be used to treat kidney injury or related diseases.⁴⁰

Conclusions

The non-contact electric fields were not harmful to the renal and liver structure of tumour-induced rats. Instead, it may optimise the renal function in normal rats.

Ethical approval

This research was carried out at the LPPT UGM and at the Animal Structure and Development Laboratory of the Faculty of Biology, UGM. LPPT UGM has been awarded ISO/IEC 17025:2000 accreditation for competence in testing and calibration.¹¹ Experimental protocol in this research was performed following approval by the Ethical Clearance Committee of LPPT UGM with ethical clearance number: 00015/4/LPPT/IV/2017, that has been previously reported.⁹ The Ethical Clearance Committee stated that this research met the ethical requirements for the study on experimental animals and that the Ethical Clearance Committee had the right to conduct monitoring during the research.

Data availability

Underlying data

Open Science Framework: Kidney and liver histology in tumour-induced rats exposed to non-contact electric fields, https://doi.org/10.17605/OSF.IO/54BYF.⁴¹

This project contains the following underlying data:

‐ Kidney and liver histological images
‐ Kidney scoring and statistical analysis
‐ Liver scoring and statistical analysis
‐ Kidney and liver charts

Extended data

Open Science Framework: Kidney and liver histology in tumour-induced rats exposed to non-contact electric fields, https://doi.org/10.17605/OSF.IO/54BYF.⁴¹

This project contains the following extended data:

‐ Ethical clearance document

Reporting guidelines

Open Science Framework: ARRIVE checklist for ‘Kidney and liver histology in tumour-induced rats exposed to non-contact electric fields’, https://doi.org/10.17605/OSF.IO/54BYF.⁴¹

Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).

Acknowledgements

Many thanks to Sugianto and Fadlil for making the device performed in this study.

References

1. Zhang Y, Ding J, Duan W, et al.: Influence of Pulsed Electromagnetic Field with Different Pulse Duty Cycles on Neurite Outgrowth in PC12 Rat Pheochromocytoma Cell. Bioelectromagn. 2005; 26: 406–411. PubMed Abstract | Publisher Full Text
2. Qiao G, Duan W, Chatwin C, et al.: Electrical Properties of Breast Cancer Cells from Impedance Measurement of Cell Suspensions. J. Phys. Conf. Ser. 2010; 224: 1–4.
3. Kirson ED, Dbaly V, Tovarys F, et al.: Alternating Electric Fields Arrest Cell Proliferation in Animal Tumor Models and Human Brain Tumors. PNAS. 2007; 104(24): 10152–10157. PubMed Abstract | Publisher Full Text | Free Full Text
4. Fu F, Xin SX, Chen W: Temperature- and Frequency-Dependent Dielectric Properties of Biological Tissues within the Temperature and Frequency Ranges Typically Used for Magnetic Resonance Imaging-Guided Focused Ultrasound Surgery. Int. J. Hyperth. 2014; 30(1): 56–65. PubMed Abstract | Publisher Full Text
5. Ji Z: Expanded Modeling of Temperature-Dependent Dielectric Properties For Microwave Thermal Ablation. Phys. Med. Biol. 2011; 56(16): 5249–5264. PubMed Abstract | Publisher Full Text | Free Full Text
6. Pop M, Molckovsky A, Chin L, et al.: Changes in Dielectric Properties at 460 kHz of Kidney and Fat During Heating: Importance for Radio-Frequency Thermal Therapy. Phys. Med. Biol. 2003; 48: 2509–2525. PubMed Abstract | Publisher Full Text
7. Porter E, Gioia AL, Santorelli A, et al.: Modeling of the Dielectric Properties of Biological Tissues within the Histology Region. IEEE Trans. Dielectr. Electr. Insul. 2017; 24(5): 3290–3301. Publisher Full Text
8. Alamsyah F, Ajrina IN, Dewi FN, et al.: Antiproliferative Effect of Electric Fields on Breast Tumor Cells In Vitro and In Vivo. Indones. J. Cancer Chemoprev. 2015; 6(3): 71–77.
9. Alamsyah F, Pratiwi R, Firdausi N, et al.: Cytotoxic T Cells Response with Decreased CD4/CD8 Ratio During Mammary Tumors Inhibition in Rats Induced by Non-Contact Electric Fields. F1000 Res. 2021; 10(35): 1–21.
10. Mujib SA, Alamsyah F, Taruno WP: Cell Death and Induced p53 Expression in Oral Cancer, HeLa, and Bone Marrow Mesenchyme Cells Under the Exposure to Noncontact Electric Fields. Integ. Med. Int. 2017; 4: 161–170. Publisher Full Text
11. Pratiwi R, Antara NY, Fadliansyah LG, et al.: CCL2 and IL18 Expressions May Associate with the Anti-Proliferative Effect of Noncontact Electro Capacitive Cancer Therapy In Vivo. F1000 Res. 2019; 8(1770): 1–14.
12. Yang M, Brackenbury WJ: Membrane Potential and Cancer Progression. Front. Physiol. 2013; 4(185): 1–10.
13. Kadir LA, Stacey M, Jolley RB: Emerging Roles of the Membrane Potential: Action Beyond the Action Potential. Front. Physiol. 2018; 9(1661): 1–10.
14. Bray F, Ferlay J, Soerjomataram I, et al.: Global Cancer Statistics 2018: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J. Clin. 2018; 68: 394–424. PubMed Abstract | Publisher Full Text
15. Costa E, Ferreira-Gonçalves T, Chasqueira G, et al.: Experimental Models as Refined Translational Tools for Breast Cancer Research. Sci. Pharm. 2020; 88(32): 1–29.
16. Abbasalipourkabir R, Dehghan A, Salehzadeh A, et al.: Induction of mammary gland tumor in female Sprague Dawley rats with LA7 cells. Afr. J. Biotechnol. 2010; 9(28): 4491–4498.
17. Akla B, Monteil J, Paraf F, et al.: A New Orthotopic Model of Human Breast Cancer in Immunocompetent Rats. Anticancer Res. 2003; 23(5A): 3761–3766. PubMed Abstract
18. Bancroft JD, Cook HC: Manual of Histology Techniques and Their Diagnostic Application. London:Churchill Livingstone;1994.
19. Gibson-Corley KN, Olivier AK, Meyerholz DK: Principles for Valid Histopathologic Scoring in Research. Vet. Pathol. 2013; 50(6): 1–22.
20. Khalid U, Pino-Chavez G, Nesargikar P, et al.: Kidney Ischaemia Reperfusion Injury in the Rat: The EGTI Scoring System as a Valid and Reliable Tool for Histological Assessment. J. Histo. Histopathol. 2016; 3(1): 1–7. Publisher Full Text
21. El-Gerbed MSA: Silymarin, Protects Against 7,12Dimethyl-benz [A] anthracene–Induced Hepatotoxicity in Albino Rats. RJPBCS. 2013; 4(3): 1534–1548.
22. Elbaz A, Ghonimi WAM: Exposure Effects of 50 Hz, 1 Gauss Magnetic Field on the Histoarchitecture Changes of Liver, Testis and Kidney of Mature Male Albino Rats. J. Cytol. Histol. 2015; 06(4): 1–6. Publisher Full Text
23. Duarte R, Jesus P, Farinha R, et al.: Adipose Tissue and Liver in DMBA Experimental Intoxication. Exp. Pathol. Health Sci. 2016; 8(1): 59:66.
24. Kotyk T, Dey N, Ashour AS, et al.: Measurement of the Glomerulus Diameter and Bowman’s Space Thickness of Renal Albino Rats. Comput. Methods Prog. Biomed. 2016; 126: 143–153. PubMed Abstract | Publisher Full Text
25. Paliwal R, Sharma V, Pracheta, et al.: Anti-Nephrotoxic Effect of Administration of Moringa oleifera Lam in Amelioration of DMBA-Induced Renal Carcinogenesis in Swiss Albino Mice. Biol. Med. 2011; 3(2): 27–35.
26. Lee JS, Ahn SS, Jung KC, et al.: Effects of 60 Hz Electromagnetic Field Exposure on Testicular Germ Cell Apoptosis in Mice. Asian J. Androl. 2004; 6(1): 29–34. PubMed Abstract
27. Sharma V, Paliwal R: Chemo Protective Role of Moringa oleifera and Its Isolated Saponin Against DMBA Induced Tissue Damage in Male Mice: A Histopathological Analysis. Int. J. Drug Dev. Res. 2012; 4(4): 215–228.
28. Rems L, Kasimova MA, Testa I, et al.: Pulsed Electric Fields Can Create Pores in the Voltage Sensors of Voltage-Gated Ion Channels. Biophys. J. 2020; 119: 190–205. PubMed Abstract | Publisher Full Text | Free Full Text
29. Petri AK, Schmiedchen K, Stunder D, et al.: Biological Effects of Exposure to Static Electric Fields in Humans and Vertebrates: A Systematic Review. Environ. Health. 2017; 16(41): 1–23.
30. Sharma V, Paliwal R, Janmeda P, et al.: Renoprotective Effects of Moringa oleifera Pods in 7,12-Dimethylbenz [a]anthracene-Exposed Mice. J. Chin. Integr. Med. 2012; 10(10): 1171–1178. Publisher Full Text
31. Neal RE, Garcia PA, Kavnoudias H, et al.: In Vivo Irreversible Electroporation Kidney Ablation: Experimentally Correlated Numerical Models. IEEE Trans. Biomed. Eng. 2015; 62(2): 561–569. Publisher Full Text
32. Cachofeiro V, Goicochea M, Vinuesa SG, et al.: Oxidative Stress and Inflammation, A Link Between Chronic Kidney Disease and Cardiovascular Disease. Kidney Int. 2008; 74: S4–S9. Publisher Full Text
33. Li N, Karin M: Is NF-kappaB the Sensor of Oxidative Stress? FASEB J. 1999; 13(10): 1137–1143. Publisher Full Text
34. Kandeel M, Abdelaziz I, Elhabashy N, et al.: Nephrotoxicity and Oxidative Stress of Single Large Dose or Two Divided Doses of Gentamicin in Rats. Pak. J. Biol. Sci. 2011; 14: 627–633. Publisher Full Text
35. Kesba HH, El-Belagi HS: Biochemical Changes in Grape Rootstocks Resulted from Humic Acid Treatments in Relation to Nematode Infection. Asian Pac. J. Trop. Biomed. 2012; 2(4): 287–293. Publisher Full Text
36. Bharali R, Tabassum J, Azad MRH: Chemomodulatory Effect of Moringa oleifera, Lam, on Hepatic Carcinogen Metabolising Enzymes, Antioxidant Parameters and Skin Papillomagenesis in Mice. Asian Pac. J. Cancer Prev. 2003; 4(2): 131–139. PubMed Abstract
37. Koul A, Arora N, Tanwar L: Lycopene Mediated Modulation of 7,12 Dimethlybenz(A) anthracene Induced Hepatic Clastogenicity in Male Balb/c Mice. Nutr. Hosp. 2010; 25(2): 304–310. PubMed Abstract
38. de Oliveira KD , Avanzo GU, Tedardi MV, et al.: Chemical Carcinogenesis by DMBA (7,12-Dimethylbenzanthracene) in Female BALB/c Mice: New Facts. Braz. J. Vet. Res. Anim. Sci. 2015; 52(2): 125–133. Publisher Full Text
39. Gutierrez G, Reines HD, Wulf-Gutierrez ME: Clinical Review: Hemorrhagic Shock. Crit. Care. 2004; 8: 373–381. PubMed Abstract | Publisher Full Text | Free Full Text
40. Chen W, Wang L, Liang P, et al.: Reducing ischemic kidney injury through application of a synchronization modulation electric field to maintain Na⁺/K⁺-ATPase functions. Sci. Transl. Med. 2022; 14(635): eabj4906. Publisher Full Text
41. Alamsyah F: Kidney and Liver Histology in Tumour-induced Rats Exposed to Non-contact Electric Fields. [Dataset]. OSF. 2022 October 22. Publisher Full Text

Comments on this article Comments (0)

Version 6

VERSION 6 PUBLISHED 01 Feb 2023

Author details Author details

Nisrina Firdausi
Roles: Data Curation, Formal Analysis, Investigation, Methodology, Resources, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Subekti Evi Dwi Nugraheni
Roles: Data Curation, Formal Analysis, Investigation, Methodology, Resources, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Ahmad Ghitha Fadhlurrahman
Roles: Investigation, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing

Luthfi Nurhidayat
Roles: Methodology, Resources, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

Rarastoeti Pratiwi
Roles: Conceptualization, Investigation, Methodology, Resources, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

Warsito Purwo Taruno
Roles: Funding Acquisition, Resources, Writing – Review & Editing

Competing interests

All authors had financial support as a research grant from Ctech Labs Edwar Technology to complete the submitted work; FA and WPT received salary from Ctech Labs Edwar Technology; NF, SEDN and AGF received honorarium from Ctech Labs Edwar Technology; no financial relationships with any organisations that might have interests in the submitted work; no other relationships or activities that could appear to have influenced the submitted work.

Grant information

This study was funded by Ctech Labs Edwar Technology.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Article Versions (6)

version 6

Revised

Published: 11 Feb 2025, 12:117

https://doi.org/10.12688/f1000research.110080.6

version 5

Revised

Published: 22 Nov 2024, 12:117

https://doi.org/10.12688/f1000research.110080.5

version 4

Revised

Published: 08 Jan 2024, 12:117

https://doi.org/10.12688/f1000research.110080.4

version 3

Revised

Published: 26 Oct 2023, 12:117

https://doi.org/10.12688/f1000research.110080.3

version 2

Revised

Published: 25 May 2023, 12:117

https://doi.org/10.12688/f1000research.110080.2

version 1

Published: 01 Feb 2023, 12:117

https://doi.org/10.12688/f1000research.110080.1

© 2023 Alamsyah F 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.

Download

Export To

metrics

	Views	Downloads
F1000Research	-	-
PubMed Central Data from PMC are received and updated monthly.	-	-

Citations

SEE MORE DETAILS

CITE

how to cite this article

Alamsyah F, Firdausi N, Nugraheni SED et al. Kidney and liver histology in tumour-induced rats exposed to non-contact electric fields [version 1; peer review: 1 approved, 1 not approved]. F1000Research 2023, 12:117 (https://doi.org/10.12688/f1000research.110080.1)

NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.

track

receive updates on this article

Track an article to receive email alerts on any updates to this article.

Open Peer Review

Version 1

VERSION 1

PUBLISHED 01 Feb 2023

Views

Reviewer Report 02 Mar 2023

Michael Staelens, Instituto de Física Corpuscular (IFIC), Paterna, Valencia, Spain

Not Approved

https://doi.org/10.5256/f1000research.121655.r162014

The manuscript, “Kidney and liver histology in tumour-induced rats exposed to non-contact electric fields”, by Alamsyah et al., reports new results on the effects of 100 kHz low-intensity non-contact electric fields on the histological profiles of kidney and liver samples taken from Sprague Dawley rats exposed to such fields (with and without tumour induction). Alternating electric fields with these particular parameters (low intensity and intermediate frequency) have become topical in oncology, especially over the last decade, due to the earlier discovery that these fields (also known as TTFields) greatly hinder the division of cancer cells. Several successful clinical trials on treating different types of cancer with TTFields have led to the subsequent FDA-approval of TTFields therapy for various cancers, such as glioblastoma. Consequently, many in vivo and in vitro studies on the efficacy of alternating electric field therapy have been conducted; however, safety studies are lacking in number and should grow in tandem with the efficacy studies. Thus, the study reported in the manuscript by Alamsyah et al. is both interesting and valuable to the scientific community (and a cancer research audience in particular); however, substantial major issues with the manuscript make the conclusions and, ultimately, the value and impact of the study uncertain. While I do believe the study has potential, the authors must revise the manuscript substantially before it should be indexed. Particularly, the literature review with regards to the Introduction and Discussion sections needs to be revisited and refocused; the statistical analysis used to determine the significance of the results does not apply, and a new analysis is required; and the very limited and overly general conclusions that do not appear to follow from the results and discussion presented in the manuscript must be stated more clearly to accurately reflect the results of the study. A complete list of my comments and concerns that must be addressed, both general and specific, are provided below.

General Comments:

There are many problems with the writing regarding grammar, punctuation, missing spaces, article usage problems, misspelled words, incorrect verb forms, faulty tense sequences, incomplete sentences, and several other sentences that simply do not make sense. In aggregate, these errors severely hinder the readability and clarity of the manuscript. I strongly recommend that the authors consider sending the manuscript to a professional English editing service, or at the very least, use one of the numerous free grammar and writing tools available online to assist with correcting many of the writing issues.
The manuscript mixes both U.S. and U.K. spellings, e.g., “tumor” and “tumour” are both used, as are “acclimatization” and “randomisation” (z vs. s). Please maintain one choice consistently in your writing.
The manuscript lacks coherence and does not communicate a cohesive story. Here are some examples of inconsistencies that negatively affect the cohesion:

- In the “Results” part of the Abstract, it reads, “The damages in the kidney and liver caused by the exposure were not significant.”, but then in the Discussion section, the following is stated “Since the significant damages of the glomerulus were observed in the kidneys of the placebo (NIT)…”, followed by, “both DMBA administration and exposure to non-contact electric fields affected the thickening of Bowman’s capsule”, which certainly sounds like there were significant damages observed in the kidney samples taken from the group that only received exposure to the electric fields (NIT).

- The need for studying the effects of electric fields-based cancer therapies on healthy tissues was motivated in the Introduction by pointing out that there might be interactions between the kidney/liver and electrostatic waves; however, the electric fields-based cancer therapies referenced and discussed in the manuscript (and that are actually used in a clinical setting) do not employ static fields, but rather alternating electric fields.

- Several of the references cited in the Discussion section that are discussed with regards to the interpretation of the results studied vastly different parameters for the electric fields than those employed in the experiments reported in the manuscript and that are typically used to treat cancers with alternating electric field therapy (e.g., 575 V/cm in one of the studies cited vs. the low-intensity of only a few V/cm maximum for alternating electric field therapy).
The manuscript often mentions the significance of results/effects; however, none of these statements include the supporting quantitative results that suggest the differences obtained are, in fact, significant (or not). Consequently, the level of significance associated with these statements is also not clear to the reader (without reading through the data files provided alongside the manuscript). Please revise all these statements to include the quantitative results of the statistical analyses that support the stated significances. For example, the following sentence on page 5, “The main damage found in the kidney glomerular was the thickening of the Bowman capsule whose scores were significant in all treatment groups (1.12±0.56 for NIT, 1.16±0.74 for INT, and 1.24±0.59 for IT groups) compared to the control (NINT) group (0.88±0.56).”, should be revised to include the corresponding H-values (or p-values, etc.) that supports the statement that these differences between scores were significant (“in all treatment groups compared to the control group”). This makes it both clear to the reader that the differences between groups are indeed significant based on the statistical analysis results obtained and provides the reader with the quantitative results, so they also get a sense of how significant (or insignificant) each particular result is without having to dig through spreadsheets of data while reading the article.
The quality of the plots provided in Figures 2 and 4 is quite low. Additionally, the dots used to represent the data are very small and difficult to distinguish (when reading the article on a tablet at 100% magnification). Please consider reproducing these plots at a higher quality (600 DPI or greater) and with the points used to represent the data enlarged and spread out more clearly, so the individual points are distinguishable without needing to zoom in (which also leads to substantial blurring due to the low quality of the plots).

Specific Comments:
Major concerns:

The data analysis section states that the scoring results were analyzed statistically using the Kruskal–Wallis test; however, the scoring results were obtained for four different groups that differed independently in two ways (i.e., there were two independent variables involved—tumour induction (or not) and treatment (or not)). Consequently, the Kruskal–Wallis test, which is only applicable in the one-factor case, cannot be used. Due to this problem, the actual statistical significance of the obtained differences between the various groups studied is not clear, and the soundness of the conclusions drawn is uncertain. If you do require a non-parametric rank-based method, then consider the Scheirer–Ray–Hare extension of the Kruskal–Wallis test, which can be used in the case where a measure may be affected by two or more factors. Alternately, the (two-factor) aligned rank transform analysis of variance (ART-ANOVA) may also be suitable.
Moreover, the conclusions do not appear to be clearly supported by the results. This concern is not only due to the previous major comment noted but also due to the following:

- The first conclusion drawn is that “The non-contact electric fields were not harmful to the renal and liver structure of tumour-induced rats"; however, one of the outcomes described in the Results and Discussion sections was that the non-contact low-intensity electric fields significantly "affected the thickening of Bowman's capsule" (NINT versus NIT), which is apparently indicative of renal damage. Consequently, I am not sure that this general conclusion is completely supported by the results of this study.

- The second conclusion drawn is that non-contact electric fields “may optimise/increase the renal function in normal rats”. What results obtained in this study demonstrate an optimization and/or increase in renal function in normal rats exposed to low-intensity non-contact electric fields? The only discussion in the manuscript regarding renal function appears to be that provided on page 8; however, the discussion there mentions renal function impairment as a possible result of DMBA-induced inflammation and does not discuss electric fields. At the end of this paragraph, non-contact electric fields are mentioned: “non-contact electric fields decreased the number of inflammations and haemorrhages in the placebo (NIT) group, as shown in Figure 2”; however, the statistical significance of this difference is not stated. Is the decrease being mentioned here actually meaningful statistically? And if so, how statistically significant was this decrease, and how exactly does this marginal decrease in observed inflammations and haemorrhages connect to the conclusion that renal function has been optimized due to treatment with non-contact electric fields? As far as I can tell, no tests or metrics that could be used to accurately evaluate any changes in kidney and/or liver function and that would support this particular conclusion were included in the experiments and analyses reported in the manuscript.
Why were damages also exhibited in the control (NINT) group? Naively, I would not have expected this outcome, and it is not explained or discussed in the manuscript.

Due to these major concerns, I do not believe that the conclusions stated in the manuscript are clearly supported by the results.

Minor concerns:

More details regarding both the electric field used in the experiments and how it was applied should be provided for clarity and reproducibility of the study:

- How were the electric fields generated? What device was used (make, model, etc.)?

- Were the electric fields static or alternating?

- What was the duty cycle?

- Given that the rats were exposed to non-contact electric fields, I assume that the electrodes were not fixed to the skin of the rats and that they received whole-body exposure to the electric fields. Is this true? Additionally, how many electrodes were used? where were they placed/attached? and with what orientation? In other words, how many electric fields were there, were they uniform or non-uniform, and with what directionality(ies)?

Please provide this information in the manuscript.
While the exposures were being performed, were there any other sources of electromagnetic radiation in the room that could be considered a source of interference?
On page 3, the last sentence in the Introduction states, “According to our knowledge, this is the first study investigating the abnormalities in the kidney and liver under exposure to 100 kHz intermediate frequency and low-intensity non-contact electric fields”, which does appear to technically be true; however, a very similar¹ study was recently published that reports the results of analyzing histological profiles of vital organs of Sprague Dawley rats exposed to 150 kHz low-intensity non-contact electromagnetic radiation. It would be valuable and interesting to revise the discussion in the manuscript to compare and contrast your results and conclusions obtained versus those reported in this very similar study in the literature.
On page 3, a value of 18 V_pp is noted after stating that the electric fields used in the study are low intensity. The electric field intensity has units of electric potential per distance (V/m in SI units), not simply volts, which is the unit of electric potential. Please revise accordingly (including at the bottom of page 3, where the same issue occurs again).
On page 3, it is stated that “Although non-contact electric fields-based therapy has the potential to treat cancer, the safety of this kind of therapy when treating healthy tissues should be investigated. This is because injuries may occur after exposure to electric fields due to the dielectric property of the kidney and liver, which may interact with electrostatic waves. Therefore, it is important to investigate the abnormalities in the kidney and liver under exposure to electric fields during cancer treatment.” The second sentence quoted here does not connect to the previous or subsequent sentences; the electric fields used in cancer treatment that this article alludes to, so-called TTFields, are alternating electric fields (time-varying), whereas electrostatic always refers to time-invariant electric fields. Please revise.
Additionally, on page 3, the sentence “the proliferation of cancer cells was successfully inhibited under exposure to intermediate frequency and low-intensity electric fields” should be revised to read “low-intensity alternating electric fields”. This statement would also benefit from more specificity, i.e., what types of cancer cells was this inhibition demonstrated for in the studies cited here and with what parameters for the electric fields employed (frequency, intensity, duration of exposure)?
Should the y-axis label for Figure 2d read “Congestion score” instead? (To maintain consistency with the other subfigures presented in Figure 2 and with Figure 4c.)
In Figure 4, the values of mean ± SD noted in each of the subfigures use inconsistent numbers of significant digits. Specifically, the values of the uncertainties are reported with greater precision than the mean values themselves, which indicates that the errors are known more precisely than the values and is very unusual.
Figures 2 and 4 include a footnote partially describing the meaning of the labels “a”, “b”, etc. as indicating significance; however, the difference between each of these labels is not clear. The exact meaning of “a”, “b”, etc. should be clarified in the notes provided under Figures 2 and 4. Additionally, while it is clear that, for example, in Figure 2a, the NINT group has the label “a” to denote that it is significant, it is not clear what it is being compared to, i.e., significant compared to which group(s)? Lastly, please change “different words a, b, c” to read “different letters” or “different labels”.
In the second paragraph of the Discussion on page 7, it is mentioned that “some biological effects of exposure to electric fields (0.6 and 340 kV/m) were revealed in humans and vertebrates, but no histological abnormalities were found in the organs, including the kidneys.” Comparing with this study does not make much sense for the following reasons:

- The particular study cited here evaluated the biological effects of static electric fields, which are not the same as the alternating electric fields exploited in cancer therapy, and that the manuscript purportedly studies the effects of.

- The parameters stated (0.6 and 340 kV/m) corresponding to this particular study that is being referenced are NOT consistent with the low-intensity electric fields used in cancer therapy (approx. 1–3 V/cm) and, again, that the manuscript appears to be studying.
The abbreviation ECCT is not defined anywhere in the manuscript.
The first sentence in the last paragraph of the Discussion section (page 9) does not make sense. Specifically, what does “Based on the evidence for the efficacy and safety of normal tissues and organs...” mean? I believe that rather than describing evidence on the “efficacy and safety of a normal tissue”, what is trying to be said here is something like, “Based on the evidence for the efficacy and safety of non-contact low-intensity electric fields regarding normal tissues and organs...”, or something along these lines. Please rephrase this sentence accordingly.
The statement regarding the role of the funders, “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”, should also mention the role of the funders regarding the interpretation of the data/results.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

No

References

1. Sundaram V, Mohammed S, Cockburn BN, Srinivasan MR, et al.: Effects of Intermediate Frequency (150 kHz) Electromagnetic Radiation on the Vital Organs of Female Sprague Dawley Rats.Biology (Basel). 2023; 12 (2). PubMed Abstract | Publisher Full Text

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Electromagnetic interactions with biological systems; microtubules; biophysics; high energy physics; particle physics.

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

CITE

Report a concern

Author Response 23 Mar 2023

Firman Alamsyah, Center for Medical Physics and Cancer Research, Ctech Labs Edwar Technology, Tangerang, 15143, Indonesia

23 Mar 2023

Author Response

We thank you for all the comments and suggestions and we will revise our paper. Below are our answers to the comments and suggestions provided in order.

General comments
... Continue reading We thank you for all the comments and suggestions and we will revise our paper. Below are our answers to the comments and suggestions provided in order.

General comments
1. We have used a professional English editing service (PaperTrue) before submitting this article. We will do one more proofreading.

2. We will use UK spelling in our revised article.

3. We will revise the abstract so that it is coherent with the contents of the paper. We will replace some of the references in the Discussion section. Previously, we did not find any references in accordance with the study conducted.

4. In the Data Analysis section and in the Figures, we have mentioned a p<0.05 value for a significant difference among groups. We will rewrite it for each data in the Results section.

5. We will fix Figures 2 and 4.

Specific comments
Major concerns
1. We will evaluate the statistical tests performed.

2. We will revise the Conclusions section to suit the Results and Discussion sections.

3. Damage to the kidneys and liver of the rats in the control group (NINT) cannot be predicted because we have excluded rats with symptoms of illness as stated in the article. Rats were also randomly selected for each group as stated also in article.

For damage to the kidney in the NINT group, if we look at the interstitial tissue, a score below 2 indicates that there is little inflammation or hemorrhage. Inflammation is part of the activation of the immune system in response to acute or chronic kidney injury which can be caused by pathogens that enter the rat’s body (Imig & Ryan, 2013).

For damage to the liver in the NINT group, if we look at the hemorrhagic and congestion scores which are below 1, this indicates that there is little or no damage to the liver. For a cellular damage score below 2, this indicates reversible damage with less than 15% necrosis. Liver hepatocytes have many vital functions, so they can proliferate extensively, which allows efficient regeneration of the liver for reversible damage (Chen et al., 2020). In addition, the liver itself is a very vulnerable organ due to its size and is the organ most frequently injured after abdominal trauma (Bilgic et al., 2014).

Minor concerns
1. We will provide more detailed information about the electric fields used in the experiment in our revised article.

2. The experiment was carried out in a special room which only contained experimental animal cages.

3. We will revise the Discussion section using the appropriate references.

4. We will include electric fields intensity data in our revised article.

5. We will revise this section (page 3).

6. We will revise this section (page 3).

7. We will revise Figure 2d.

8. We will revise Figure 4.

9. We will revise Figures 2 and 4.

10. We will revise the Discussion section using the appropriate references.

11. We will write what ECCT stands for in our revised article.

12. We will rephrase this sentence. There may be phrases lost in the editing process.

13. We will add the statement regarding the role of the funders in the interpretation of the data/results.

References
Bilgiç I, Gelecek S, Akgün AE, et al.: Evaluation of liver injury in a tertiary hospital: a retrospective study. Ulus Travma Acil Cerrahi Derg. 2014; 20(5): 359-365.

Chen F, Jimenez RJ, Sharma K, et al.: Broad Distribution of Hepatocyte Proliferation in Liver Homeostasis and Regeneration. Cell Stem Cell. 2020; 26(1):27-33

Imig JD, Ryan MJ: Immune and Inflammatory Role in Renal Disease. Compr. Physiol. 2013; 3(2): 957-976.
We thank you for all the comments and suggestions and we will revise our paper. Below are our answers to the comments and suggestions provided in order.

General comments
1. We have used a professional English editing service (PaperTrue) before submitting this article. We will do one more proofreading.

2. We will use UK spelling in our revised article.

3. We will revise the abstract so that it is coherent with the contents of the paper. We will replace some of the references in the Discussion section. Previously, we did not find any references in accordance with the study conducted.

4. In the Data Analysis section and in the Figures, we have mentioned a p<0.05 value for a significant difference among groups. We will rewrite it for each data in the Results section.

5. We will fix Figures 2 and 4.

Specific comments
Major concerns
1. We will evaluate the statistical tests performed.

2. We will revise the Conclusions section to suit the Results and Discussion sections.

3. Damage to the kidneys and liver of the rats in the control group (NINT) cannot be predicted because we have excluded rats with symptoms of illness as stated in the article. Rats were also randomly selected for each group as stated also in article.

For damage to the kidney in the NINT group, if we look at the interstitial tissue, a score below 2 indicates that there is little inflammation or hemorrhage. Inflammation is part of the activation of the immune system in response to acute or chronic kidney injury which can be caused by pathogens that enter the rat’s body (Imig & Ryan, 2013).

For damage to the liver in the NINT group, if we look at the hemorrhagic and congestion scores which are below 1, this indicates that there is little or no damage to the liver. For a cellular damage score below 2, this indicates reversible damage with less than 15% necrosis. Liver hepatocytes have many vital functions, so they can proliferate extensively, which allows efficient regeneration of the liver for reversible damage (Chen et al., 2020). In addition, the liver itself is a very vulnerable organ due to its size and is the organ most frequently injured after abdominal trauma (Bilgic et al., 2014).

Minor concerns
1. We will provide more detailed information about the electric fields used in the experiment in our revised article.

2. The experiment was carried out in a special room which only contained experimental animal cages.

3. We will revise the Discussion section using the appropriate references.

4. We will include electric fields intensity data in our revised article.

5. We will revise this section (page 3).

6. We will revise this section (page 3).

7. We will revise Figure 2d.

8. We will revise Figure 4.

9. We will revise Figures 2 and 4.

10. We will revise the Discussion section using the appropriate references.

11. We will write what ECCT stands for in our revised article.

12. We will rephrase this sentence. There may be phrases lost in the editing process.

13. We will add the statement regarding the role of the funders in the interpretation of the data/results.

References
Bilgiç I, Gelecek S, Akgün AE, et al.: Evaluation of liver injury in a tertiary hospital: a retrospective study. Ulus Travma Acil Cerrahi Derg. 2014; 20(5): 359-365.

Chen F, Jimenez RJ, Sharma K, et al.: Broad Distribution of Hepatocyte Proliferation in Liver Homeostasis and Regeneration. Cell Stem Cell. 2020; 26(1):27-33

Imig JD, Ryan MJ: Immune and Inflammatory Role in Renal Disease. Compr. Physiol. 2013; 3(2): 957-976.
Competing Interests: No competing interests were disclosed. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 23 Mar 2023

Firman Alamsyah, Center for Medical Physics and Cancer Research, Ctech Labs Edwar Technology, Tangerang, 15143, Indonesia

23 Mar 2023

Author Response

We thank you for all the comments and suggestions and we will revise our paper. Below are our answers to the comments and suggestions provided in order.

General comments
... Continue reading We thank you for all the comments and suggestions and we will revise our paper. Below are our answers to the comments and suggestions provided in order.

General comments
1. We have used a professional English editing service (PaperTrue) before submitting this article. We will do one more proofreading.

2. We will use UK spelling in our revised article.

3. We will revise the abstract so that it is coherent with the contents of the paper. We will replace some of the references in the Discussion section. Previously, we did not find any references in accordance with the study conducted.

4. In the Data Analysis section and in the Figures, we have mentioned a p<0.05 value for a significant difference among groups. We will rewrite it for each data in the Results section.

5. We will fix Figures 2 and 4.

Specific comments
Major concerns
1. We will evaluate the statistical tests performed.

2. We will revise the Conclusions section to suit the Results and Discussion sections.

3. Damage to the kidneys and liver of the rats in the control group (NINT) cannot be predicted because we have excluded rats with symptoms of illness as stated in the article. Rats were also randomly selected for each group as stated also in article.

For damage to the kidney in the NINT group, if we look at the interstitial tissue, a score below 2 indicates that there is little inflammation or hemorrhage. Inflammation is part of the activation of the immune system in response to acute or chronic kidney injury which can be caused by pathogens that enter the rat’s body (Imig & Ryan, 2013).

For damage to the liver in the NINT group, if we look at the hemorrhagic and congestion scores which are below 1, this indicates that there is little or no damage to the liver. For a cellular damage score below 2, this indicates reversible damage with less than 15% necrosis. Liver hepatocytes have many vital functions, so they can proliferate extensively, which allows efficient regeneration of the liver for reversible damage (Chen et al., 2020). In addition, the liver itself is a very vulnerable organ due to its size and is the organ most frequently injured after abdominal trauma (Bilgic et al., 2014).

Minor concerns
1. We will provide more detailed information about the electric fields used in the experiment in our revised article.

2. The experiment was carried out in a special room which only contained experimental animal cages.

3. We will revise the Discussion section using the appropriate references.

4. We will include electric fields intensity data in our revised article.

5. We will revise this section (page 3).

6. We will revise this section (page 3).

7. We will revise Figure 2d.

8. We will revise Figure 4.

9. We will revise Figures 2 and 4.

10. We will revise the Discussion section using the appropriate references.

11. We will write what ECCT stands for in our revised article.

12. We will rephrase this sentence. There may be phrases lost in the editing process.

13. We will add the statement regarding the role of the funders in the interpretation of the data/results.

References
Bilgiç I, Gelecek S, Akgün AE, et al.: Evaluation of liver injury in a tertiary hospital: a retrospective study. Ulus Travma Acil Cerrahi Derg. 2014; 20(5): 359-365.

Chen F, Jimenez RJ, Sharma K, et al.: Broad Distribution of Hepatocyte Proliferation in Liver Homeostasis and Regeneration. Cell Stem Cell. 2020; 26(1):27-33

Imig JD, Ryan MJ: Immune and Inflammatory Role in Renal Disease. Compr. Physiol. 2013; 3(2): 957-976.
We thank you for all the comments and suggestions and we will revise our paper. Below are our answers to the comments and suggestions provided in order.

General comments
1. We have used a professional English editing service (PaperTrue) before submitting this article. We will do one more proofreading.

2. We will use UK spelling in our revised article.

3. We will revise the abstract so that it is coherent with the contents of the paper. We will replace some of the references in the Discussion section. Previously, we did not find any references in accordance with the study conducted.

4. In the Data Analysis section and in the Figures, we have mentioned a p<0.05 value for a significant difference among groups. We will rewrite it for each data in the Results section.

5. We will fix Figures 2 and 4.

Specific comments
Major concerns
1. We will evaluate the statistical tests performed.

2. We will revise the Conclusions section to suit the Results and Discussion sections.

3. Damage to the kidneys and liver of the rats in the control group (NINT) cannot be predicted because we have excluded rats with symptoms of illness as stated in the article. Rats were also randomly selected for each group as stated also in article.

For damage to the kidney in the NINT group, if we look at the interstitial tissue, a score below 2 indicates that there is little inflammation or hemorrhage. Inflammation is part of the activation of the immune system in response to acute or chronic kidney injury which can be caused by pathogens that enter the rat’s body (Imig & Ryan, 2013).

For damage to the liver in the NINT group, if we look at the hemorrhagic and congestion scores which are below 1, this indicates that there is little or no damage to the liver. For a cellular damage score below 2, this indicates reversible damage with less than 15% necrosis. Liver hepatocytes have many vital functions, so they can proliferate extensively, which allows efficient regeneration of the liver for reversible damage (Chen et al., 2020). In addition, the liver itself is a very vulnerable organ due to its size and is the organ most frequently injured after abdominal trauma (Bilgic et al., 2014).

Minor concerns
1. We will provide more detailed information about the electric fields used in the experiment in our revised article.

2. The experiment was carried out in a special room which only contained experimental animal cages.

3. We will revise the Discussion section using the appropriate references.

4. We will include electric fields intensity data in our revised article.

5. We will revise this section (page 3).

6. We will revise this section (page 3).

7. We will revise Figure 2d.

8. We will revise Figure 4.

9. We will revise Figures 2 and 4.

10. We will revise the Discussion section using the appropriate references.

11. We will write what ECCT stands for in our revised article.

12. We will rephrase this sentence. There may be phrases lost in the editing process.

13. We will add the statement regarding the role of the funders in the interpretation of the data/results.

References
Bilgiç I, Gelecek S, Akgün AE, et al.: Evaluation of liver injury in a tertiary hospital: a retrospective study. Ulus Travma Acil Cerrahi Derg. 2014; 20(5): 359-365.

Chen F, Jimenez RJ, Sharma K, et al.: Broad Distribution of Hepatocyte Proliferation in Liver Homeostasis and Regeneration. Cell Stem Cell. 2020; 26(1):27-33

Imig JD, Ryan MJ: Immune and Inflammatory Role in Renal Disease. Compr. Physiol. 2013; 3(2): 957-976.
Competing Interests: No competing interests were disclosed. Close
Report a concern

Views

Reviewer Report 27 Feb 2023

Chandran Nadarajan, School of Medical Sciences, Hospital Universiti Sains Malaysia, Kota Bharu, Malaysia

Approved

https://doi.org/10.5256/f1000research.121655.r162015

Editorial Note from F1000Research – 06/03/2023:

This report has been updated after the reviewer informed the editorial team that they had further comments after their initial review was published. The changes are due to some factors which were not ... Continue reading

Editorial Note from F1000Research – 06/03/2023:

This report has been updated after the reviewer informed the editorial team that they had further comments after their initial review was published. The changes are due to some factors which were not assessed in the initial review and were picked up when the reviewer read the article again. This update has not changed the ‘Approved’ status that the reviewer originally assigned.

This study touches on the effect of a new technology being introduced in oncology and answers some of the questions regarding the safety issue of the device. This information is critical to further utilize this device in the general public.

Some additional information from the authors could elevate the write-up:

Why do the authors choose the voltage 100kHz and 18v, specifically? Were the preliminary results referred to published? Are there any other articles supporting the usage of this voltage and frequency?
Is there any statistical analysis done? This will help strengthen the conclusion.
Why was there damage in the kidney interstitial tissue and liver damage in the NINT group?

In addition, some of the articles referred to use different voltage and frequency levels; therefore, it couldn't be a direct comparison of this study. Such correlation needed to be taken with caution.

Overall, this study tries to answer the safety aspect of this non-contact electrical field therapy and highlights some promising changes.

--------------------------------------------------------------------------

First of all, I maintain that the paper is quite impressive and constructive with a novel idea. However, I would suggest a few additional points:

Figures 1 and 3 are very small, with many icons. Please enhance them.
Figures 2, and 4 suggest changing to the Whisker box plot and being made bigger. It will be better.
Utilization of rank order, like Mann Whitney, might help to bring forward the idea better.
Conclusion is a little too short and needs to be improved. Adding limitations and further direction might help. This can be done in the discussion.
IRB date, number, and place should be shown.
In several places, language is unclear, ambiguous, or confusing. It is necessary to use a professional editing service to improve it before indexing.
I think the title "Effects of non-contact electric fields on kidney and liver histology in tumour-induced rats." Might be more appropriate.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

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

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Interventional oncology, interventional and diagnostic radiology

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.

CITE

Report a concern

Author Response 01 Mar 2023

Firman Alamsyah, Center for Medical Physics and Cancer Research, Ctech Labs Edwar Technology, Tangerang, 15143, Indonesia

01 Mar 2023

Author Response

I will answer the reviewer's comments sequentially in the order of the comments.

1. We have conducted an in vitro study using various frequencies and intensities, but this study has ... Continue reading I will answer the reviewer's comments sequentially in the order of the comments.

1. We have conducted an in vitro study using various frequencies and intensities, but this study has not been published. We used 100 kHz and 18 Vpp for in vivo study because this frequency and intensity gave the best results in in vitro study, where 28-39% of breast cancer cells died (Alamsyah et al., 2015). In the preliminary in vivo study using 9 mice, the frequency of 100 kHz and intensity of 18 Vpp also gave good results, where the tumor size was reduced by more than 67% and the results of histopathological analysis on normal skin and breast tissue, showed no damage (Alamsyah et al., 2015).

2. Yes, we did the statistical analysis and it has been written in the article in the Data Analysis section. Kidney and liver scoring data, as well as statistical analysis can be seen in the links provided in the Data Availability section of the article.

3. Damage to the kidneys and liver of the rats in the control group (NINT) cannot be predicted, because we have excluded rats with symptoms of illness as stated in the article. Rats were also randomly selected for each group as stated also in article.

For damage in the renal interstitial tissue of the NINT group, a score below 2 indicated that there was little inflammation or hemorrhage. Inflammation is part of the activation of the immune system in response to acute or chronic kidney injury which can be caused by pathogens that enter the rat’s body (Imig & Ryan, 2013).

For damage in the liver of the NINT group, if we look at the hemorrhagic and congestion scores which are below 1, this indicated that there was little or no damage to the liver. For a cellular damage score below 2, this indicated reversible damage with less than 15% necrosis. Liver hepatocytes have many vital functions, so they can proliferate extensively, which allows efficient regeneration of the liver for reversible damage (Chen et al., 2020). In addition, the liver itself is a very vulnerable organ due to its size and is the organ most frequently injured after abdominal trauma (Bilgic et al., 2014). Rats are active animals and may chase or fight each other in communal cages which can cause trauma to their body (Steimer, 2011).

4. We found no reference to kidney or liver damage at intermediate frequency and low intensity electric fields, especially 100 kHz and 18 Vpp. We have also stated this in the article. In the Introduction section, we have also stated that this is the first study to investigate abnormalities in the kidney and liver under exposure to a intermediate frequency of 100 kHz and a low intensity non-contact electric field.

We will add necessary information to our article from the answers to the reviewer's questions.

Thank you.

References
Alamsyah F, Ajrina IN, Dewi FN, et al.: Antiproliferative Effect of Electric Fields on Breast Tumor Cells In Vitro and In Vivo. Indones. J. Cancer Chemoprev. 2015; 6(3): 71–77.
Bilgiç I, Gelecek S, Akgün AE, et al.: Evaluation of liver injury in a tertiary hospital: a retrospective study. Ulus Travma Acil Cerrahi Derg. 2014; 20(5): 359-365.
Chen F, Jimenez RJ, Sharma K, et al.: Broad Distribution of Hepatocyte Proliferation in Liver Homeostasis and Regeneration. Cell Stem Cell. 2020; 26(1):27-33
Imig JD, Ryan MJ: Immune and Inflammatory Role in Renal Disease. Compr. Physiol. 2013; 3(2): 957-976.
Steimer T: Animal models of anxiety disorders in rats and mice: some conceptual issues. Trans Res. 2011; 495-506.
I will answer the reviewer's comments sequentially in the order of the comments.

1. We have conducted an in vitro study using various frequencies and intensities, but this study has not been published. We used 100 kHz and 18 Vpp for in vivo study because this frequency and intensity gave the best results in in vitro study, where 28-39% of breast cancer cells died (Alamsyah et al., 2015). In the preliminary in vivo study using 9 mice, the frequency of 100 kHz and intensity of 18 Vpp also gave good results, where the tumor size was reduced by more than 67% and the results of histopathological analysis on normal skin and breast tissue, showed no damage (Alamsyah et al., 2015).

2. Yes, we did the statistical analysis and it has been written in the article in the Data Analysis section. Kidney and liver scoring data, as well as statistical analysis can be seen in the links provided in the Data Availability section of the article.

3. Damage to the kidneys and liver of the rats in the control group (NINT) cannot be predicted, because we have excluded rats with symptoms of illness as stated in the article. Rats were also randomly selected for each group as stated also in article.

For damage in the renal interstitial tissue of the NINT group, a score below 2 indicated that there was little inflammation or hemorrhage. Inflammation is part of the activation of the immune system in response to acute or chronic kidney injury which can be caused by pathogens that enter the rat’s body (Imig & Ryan, 2013).

For damage in the liver of the NINT group, if we look at the hemorrhagic and congestion scores which are below 1, this indicated that there was little or no damage to the liver. For a cellular damage score below 2, this indicated reversible damage with less than 15% necrosis. Liver hepatocytes have many vital functions, so they can proliferate extensively, which allows efficient regeneration of the liver for reversible damage (Chen et al., 2020). In addition, the liver itself is a very vulnerable organ due to its size and is the organ most frequently injured after abdominal trauma (Bilgic et al., 2014). Rats are active animals and may chase or fight each other in communal cages which can cause trauma to their body (Steimer, 2011).

4. We found no reference to kidney or liver damage at intermediate frequency and low intensity electric fields, especially 100 kHz and 18 Vpp. We have also stated this in the article. In the Introduction section, we have also stated that this is the first study to investigate abnormalities in the kidney and liver under exposure to a intermediate frequency of 100 kHz and a low intensity non-contact electric field.

We will add necessary information to our article from the answers to the reviewer's questions.

Thank you.

References
Alamsyah F, Ajrina IN, Dewi FN, et al.: Antiproliferative Effect of Electric Fields on Breast Tumor Cells In Vitro and In Vivo. Indones. J. Cancer Chemoprev. 2015; 6(3): 71–77.
Bilgiç I, Gelecek S, Akgün AE, et al.: Evaluation of liver injury in a tertiary hospital: a retrospective study. Ulus Travma Acil Cerrahi Derg. 2014; 20(5): 359-365.
Chen F, Jimenez RJ, Sharma K, et al.: Broad Distribution of Hepatocyte Proliferation in Liver Homeostasis and Regeneration. Cell Stem Cell. 2020; 26(1):27-33
Imig JD, Ryan MJ: Immune and Inflammatory Role in Renal Disease. Compr. Physiol. 2013; 3(2): 957-976.
Steimer T: Animal models of anxiety disorders in rats and mice: some conceptual issues. Trans Res. 2011; 495-506.
Competing Interests: No competing interests were disclosed. Close
Report a concern
Author Response 21 Mar 2023

Firman Alamsyah, Center for Medical Physics and Cancer Research, Ctech Labs Edwar Technology, Tangerang, 15143, Indonesia

21 Mar 2023

Author Response

We thank you for the additional suggestions and we will revise our paper. Below are our answers to the suggestions provided in order.

1. We will enhance Figure 1 ... Continue reading We thank you for the additional suggestions and we will revise our paper. Below are our answers to the suggestions provided in order.

1. We will enhance Figure 1 and Figure 3 in our revision.

2. We will consider to use the Whisker box plot in Figure 2 and Figure 4.

3. We have conducted Mann-Whitney test (α=0.05) in our statistical analysis.

4. We will improve the conclusions of our study and we will discuss the limitations of this study. We have written further directions of this study at the end of the discussion section.

5. IRB date, number, and place have been written in the ethical approval section.

6. We have used a professional editing service (PaperTrue) before submitting this article. We will do one more proofreading.

7. We will improve the title of this article.
We thank you for the additional suggestions and we will revise our paper. Below are our answers to the suggestions provided in order.

1. We will enhance Figure 1 and Figure 3 in our revision.

2. We will consider to use the Whisker box plot in Figure 2 and Figure 4.

3. We have conducted Mann-Whitney test (α=0.05) in our statistical analysis.

4. We will improve the conclusions of our study and we will discuss the limitations of this study. We have written further directions of this study at the end of the discussion section.

5. IRB date, number, and place have been written in the ethical approval section.

6. We have used a professional editing service (PaperTrue) before submitting this article. We will do one more proofreading.

7. We will improve the title of this article.
Competing Interests: No competing interests were disclosed. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 01 Mar 2023

Firman Alamsyah, Center for Medical Physics and Cancer Research, Ctech Labs Edwar Technology, Tangerang, 15143, Indonesia

01 Mar 2023

Author Response

I will answer the reviewer's comments sequentially in the order of the comments.

1. We have conducted an in vitro study using various frequencies and intensities, but this study has ... Continue reading I will answer the reviewer's comments sequentially in the order of the comments.

1. We have conducted an in vitro study using various frequencies and intensities, but this study has not been published. We used 100 kHz and 18 Vpp for in vivo study because this frequency and intensity gave the best results in in vitro study, where 28-39% of breast cancer cells died (Alamsyah et al., 2015). In the preliminary in vivo study using 9 mice, the frequency of 100 kHz and intensity of 18 Vpp also gave good results, where the tumor size was reduced by more than 67% and the results of histopathological analysis on normal skin and breast tissue, showed no damage (Alamsyah et al., 2015).

2. Yes, we did the statistical analysis and it has been written in the article in the Data Analysis section. Kidney and liver scoring data, as well as statistical analysis can be seen in the links provided in the Data Availability section of the article.

3. Damage to the kidneys and liver of the rats in the control group (NINT) cannot be predicted, because we have excluded rats with symptoms of illness as stated in the article. Rats were also randomly selected for each group as stated also in article.

For damage in the renal interstitial tissue of the NINT group, a score below 2 indicated that there was little inflammation or hemorrhage. Inflammation is part of the activation of the immune system in response to acute or chronic kidney injury which can be caused by pathogens that enter the rat’s body (Imig & Ryan, 2013).

For damage in the liver of the NINT group, if we look at the hemorrhagic and congestion scores which are below 1, this indicated that there was little or no damage to the liver. For a cellular damage score below 2, this indicated reversible damage with less than 15% necrosis. Liver hepatocytes have many vital functions, so they can proliferate extensively, which allows efficient regeneration of the liver for reversible damage (Chen et al., 2020). In addition, the liver itself is a very vulnerable organ due to its size and is the organ most frequently injured after abdominal trauma (Bilgic et al., 2014). Rats are active animals and may chase or fight each other in communal cages which can cause trauma to their body (Steimer, 2011).

4. We found no reference to kidney or liver damage at intermediate frequency and low intensity electric fields, especially 100 kHz and 18 Vpp. We have also stated this in the article. In the Introduction section, we have also stated that this is the first study to investigate abnormalities in the kidney and liver under exposure to a intermediate frequency of 100 kHz and a low intensity non-contact electric field.

We will add necessary information to our article from the answers to the reviewer's questions.

Thank you.

References
Alamsyah F, Ajrina IN, Dewi FN, et al.: Antiproliferative Effect of Electric Fields on Breast Tumor Cells In Vitro and In Vivo. Indones. J. Cancer Chemoprev. 2015; 6(3): 71–77.
Bilgiç I, Gelecek S, Akgün AE, et al.: Evaluation of liver injury in a tertiary hospital: a retrospective study. Ulus Travma Acil Cerrahi Derg. 2014; 20(5): 359-365.
Chen F, Jimenez RJ, Sharma K, et al.: Broad Distribution of Hepatocyte Proliferation in Liver Homeostasis and Regeneration. Cell Stem Cell. 2020; 26(1):27-33
Imig JD, Ryan MJ: Immune and Inflammatory Role in Renal Disease. Compr. Physiol. 2013; 3(2): 957-976.
Steimer T: Animal models of anxiety disorders in rats and mice: some conceptual issues. Trans Res. 2011; 495-506.
I will answer the reviewer's comments sequentially in the order of the comments.

1. We have conducted an in vitro study using various frequencies and intensities, but this study has not been published. We used 100 kHz and 18 Vpp for in vivo study because this frequency and intensity gave the best results in in vitro study, where 28-39% of breast cancer cells died (Alamsyah et al., 2015). In the preliminary in vivo study using 9 mice, the frequency of 100 kHz and intensity of 18 Vpp also gave good results, where the tumor size was reduced by more than 67% and the results of histopathological analysis on normal skin and breast tissue, showed no damage (Alamsyah et al., 2015).

2. Yes, we did the statistical analysis and it has been written in the article in the Data Analysis section. Kidney and liver scoring data, as well as statistical analysis can be seen in the links provided in the Data Availability section of the article.

3. Damage to the kidneys and liver of the rats in the control group (NINT) cannot be predicted, because we have excluded rats with symptoms of illness as stated in the article. Rats were also randomly selected for each group as stated also in article.

For damage in the renal interstitial tissue of the NINT group, a score below 2 indicated that there was little inflammation or hemorrhage. Inflammation is part of the activation of the immune system in response to acute or chronic kidney injury which can be caused by pathogens that enter the rat’s body (Imig & Ryan, 2013).

For damage in the liver of the NINT group, if we look at the hemorrhagic and congestion scores which are below 1, this indicated that there was little or no damage to the liver. For a cellular damage score below 2, this indicated reversible damage with less than 15% necrosis. Liver hepatocytes have many vital functions, so they can proliferate extensively, which allows efficient regeneration of the liver for reversible damage (Chen et al., 2020). In addition, the liver itself is a very vulnerable organ due to its size and is the organ most frequently injured after abdominal trauma (Bilgic et al., 2014). Rats are active animals and may chase or fight each other in communal cages which can cause trauma to their body (Steimer, 2011).

4. We found no reference to kidney or liver damage at intermediate frequency and low intensity electric fields, especially 100 kHz and 18 Vpp. We have also stated this in the article. In the Introduction section, we have also stated that this is the first study to investigate abnormalities in the kidney and liver under exposure to a intermediate frequency of 100 kHz and a low intensity non-contact electric field.

We will add necessary information to our article from the answers to the reviewer's questions.

Thank you.

References
Alamsyah F, Ajrina IN, Dewi FN, et al.: Antiproliferative Effect of Electric Fields on Breast Tumor Cells In Vitro and In Vivo. Indones. J. Cancer Chemoprev. 2015; 6(3): 71–77.
Bilgiç I, Gelecek S, Akgün AE, et al.: Evaluation of liver injury in a tertiary hospital: a retrospective study. Ulus Travma Acil Cerrahi Derg. 2014; 20(5): 359-365.
Chen F, Jimenez RJ, Sharma K, et al.: Broad Distribution of Hepatocyte Proliferation in Liver Homeostasis and Regeneration. Cell Stem Cell. 2020; 26(1):27-33
Imig JD, Ryan MJ: Immune and Inflammatory Role in Renal Disease. Compr. Physiol. 2013; 3(2): 957-976.
Steimer T: Animal models of anxiety disorders in rats and mice: some conceptual issues. Trans Res. 2011; 495-506.
Competing Interests: No competing interests were disclosed. Close
Report a concern
Author Response 21 Mar 2023

Firman Alamsyah, Center for Medical Physics and Cancer Research, Ctech Labs Edwar Technology, Tangerang, 15143, Indonesia

21 Mar 2023

Author Response

We thank you for the additional suggestions and we will revise our paper. Below are our answers to the suggestions provided in order.

1. We will enhance Figure 1 ... Continue reading We thank you for the additional suggestions and we will revise our paper. Below are our answers to the suggestions provided in order.

1. We will enhance Figure 1 and Figure 3 in our revision.

2. We will consider to use the Whisker box plot in Figure 2 and Figure 4.

3. We have conducted Mann-Whitney test (α=0.05) in our statistical analysis.

4. We will improve the conclusions of our study and we will discuss the limitations of this study. We have written further directions of this study at the end of the discussion section.

5. IRB date, number, and place have been written in the ethical approval section.

6. We have used a professional editing service (PaperTrue) before submitting this article. We will do one more proofreading.

7. We will improve the title of this article.
We thank you for the additional suggestions and we will revise our paper. Below are our answers to the suggestions provided in order.

1. We will enhance Figure 1 and Figure 3 in our revision.

2. We will consider to use the Whisker box plot in Figure 2 and Figure 4.

3. We have conducted Mann-Whitney test (α=0.05) in our statistical analysis.

4. We will improve the conclusions of our study and we will discuss the limitations of this study. We have written further directions of this study at the end of the discussion section.

5. IRB date, number, and place have been written in the ethical approval section.

6. We have used a professional editing service (PaperTrue) before submitting this article. We will do one more proofreading.

7. We will improve the title of this article.
Competing Interests: No competing interests were disclosed. Close
Report a concern

Comments on this article Comments (0)

Version 6

VERSION 6 PUBLISHED 01 Feb 2023

Open Peer Review

Reviewer Status

Reviewer Reports

	Invited Reviewers
	1	2	3	4
Version 6 (revision) 11 Feb 25				read
Version 5 (revision) 22 Nov 24				read
Version 4 (revision) 08 Jan 24			read
Version 3 (revision) 26 Oct 23		read
Version 2 (revision) 25 May 23		read
Version 1 01 Feb 23	read	read

Chandran Nadarajan, Hospital Universiti Sains Malaysia, Kota Bharu, Malaysia
Michael Staelens, Instituto de Física Corpuscular (IFIC), Paterna, Spain
Tejaswi Worlikar, University of Michigan, Ann Arbor, USA
Mohamed Hisham Fouad Aref, Biomedical Engineering Researcher, Cairo, Egypt

Comments on this article

All Comments(0)

Add a comment

Browse by related subjects

Back to all reports

Reviewer Report

4 Views

19 Feb 2025 | for Version 6

Mohamed Hisham Fouad Aref, Biomedical Engineering Researcher, Cairo, Egypt

4 Views Cite this report Responses(0)

Approved

Dear Editor and Authors,
I really appreciate all the hard work done by the Authors to edit and enhance their article,
looking forward to see their work soon.
Best regards.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Biomedical Engineering, Hyperbaric Oxygenation, Biomedical Enhancement, Biomedical Applications, Hyperspectral Image Analysis

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.

Respond to this report

Responses (0)

Back to all reports

Reviewer Report

9 Views

31 Dec 2024 | for Version 5

Mohamed Hisham Fouad Aref, Biomedical Engineering Researcher, Cairo, Egypt

9 Views Cite this report Responses(1)

Approved With Reservations

The article is presenting one of the interesting subjects related to kidney and liver tumors. Additionally, the authors did a great job to enhance their previous versions. However, I have thoroughly reviewed the article, the article still needs some additional editing and enhancement to be ready for next step and indexing. The following aspects of the paper should be addressed for improvement:

The current title is specific, clearly indicating the study's focus on the effects of non-contact electric fields on kidney and liver histology in tumor-induced rats. However, It would benefit from revision by the authors to make it shorter, more concise, and informative, capturing the reader’s attention with clear and appealing language.
The authors are preferred to add “highlights bullets” to elaborate the importance of their research and grab the reader`s attention.
The “Abstract” section it needs to be revised by the authors and re-editing in a better presentable manner without skipping the ideas to comply with the presented idea of the manuscript and more organized.
Kindly in the “Abstract” section have several week points which needs to be enhanced, such as:

It lacks detail on the sample size and the extent of variability in observed results.
Phrases like “safe in some contexts” are vague and could benefit from precise language to avoid ambiguity.

The “Introduction” section thorough background on the potential of non-contact electric field therapy (ECCT) and its relevance in cancer treatment. However, it still needs to be revised and enhanced in each of the following:

Repetition of points about electric field effects (e.g., intermediate-frequency benefits) could have been condensed.
Does not clearly state the specific hypothesis or expected outcomes of the study.
Lacks detailed justification for the chosen frequency (100 kHz) compared to previously studied 150 kHz.

Kindly read some of these useful previous researches with different and various techniques toward your study (Tumor detection) which could be helpful in your “Introduction and Literature sections” for your research outcome and could be more valuable to your research, feel free to use them in your study or not after reading them:

Zhang X, et al., 2024 (Ref 1)
Matos LC, et al., 2021 (Ref 2)
Aref MH, et al., 2024 (Ref 3)
Sequin, Emily Katherine. Effects of induced electric fields on tissues and cells. Diss. The Ohio State University, 2014, https://etd.ohiolink.edu/acprod/odb_etd/etd/r/1501/10?clear=10&p10_accession_num=osu1403869854.
Rijal, Nava P. Wireless non-contact electric field therapy improves healing in a porcine model. Diss. University of Cincinnati, 2021https://www.proquest.com/openview/a8b24b1ee8cb580713289e3734531643/1?pq-origsite=gscholar&cbl=18750&diss=y.

The “Materials & Methods section is detailed, with clear descriptions of experimental design, animal care, and histological procedures. On the other hand, the section needs to be revised and re-edit regarding the following recommendations:

Limited sample size (2 kidneys and livers per group) raises concerns about the statistical power and generalizability of results.
It is unclear why data from other animals in each group were not included, which undermines transparency.
Does not provide justification for the specific exposure duration (10 hours daily for 21 days) or how this relates to potential clinical applications.

The “Result” section is presented with statistical analysis, including p-values for differences between groups. However, it needs the following:

Key findings, such as the reduction of kidney inflammation and liver hemorrhage, are not adequately contextualized with broader implications.
Some results, such as the lack of fibrosis or chronic congestion, are mentioned but not sufficiently explained.
Limited discussion of variability in results across groups, which could indicate inconsistencies.

The “Discussion” section provides a logical interpretation of findings, linking observed changes to potential mechanisms (e.g., oxidative stress, immune responses). For improvement, kindly see the following recommendations:

Overemphasis on certain findings (e.g., kidney safety) while underexploring significant liver damage in healthy rats.
Speculative claims, such as "this therapy may be used to treat kidney injuries," lack experimental validation.
The discussion of ECCT parameters (frequency, intensity) is insufficient; no clear rationale is given for choosing these specific conditions.
Does not address the broader implications of findings for clinical translation, such as potential risks in non-cancer patients.
Combine previous researches toward your study and compared it with your findings, feel free to use other techniques to compare your study`s technique.

Kindly revise the “Reference” section and update it, to cover your study`s background and literature review. Additionally, to be in the latest and nearest year to your research, as some reference needs to be replaced with the updated researches, such as: (Ref #1-2005 / Ref #2-2010 / Ref #3-2007 / Ref #6-2003/ Ref #18-2010 / Ref #19-2003…etc.).
The English language in this manuscript requires considerable improvement, as it contains several typographical errors and overly long sentences with mixed ideas, which reduce clarity and impact. I recommend the authors revise the manuscript with more concise and well-structured sentences.

In conclusion, I commend the authors for their hard work and efforts in preparing this manuscript. However, I believe further refinement is needed to improve the organization of ideas and to more clearly present the study's findings with additional detail and clearer illustrations.
Best regards and good luck,

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

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

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Yes

References

1. Zhang X, Yan Z, Huang L, Yu X, et al.: A new tumor-treating device OM-100 with low-frequency magnetic fields inhibits proliferation and metastasis in liver cancer.BMC Cancer. 2024; 24 (1): 1383 PubMed Abstract | Publisher Full Text
2. Matos LC, Machado JP, Monteiro FJ, Greten HJ: Perspectives, Measurability and Effects of Non-Contact Biofield-Based Practices: A Narrative Review of Quantitative Research.Int J Environ Res Public Health. 2021; 18 (12). PubMed Abstract | Publisher Full Text
3. Aref MH, Korganbayev S, Aboughaleb IH, Hussein AA, et al.: Custom Hyperspectral Imaging System Reveals Unique Spectral Signatures of Heart, Kidney, and Liver Tissues.Spectrochim Acta A Mol Biomol Spectrosc. 2024; 305: 123363 PubMed Abstract | Publisher Full Text

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Biomedical EngineeringHyperbaric OxygenationBiomedical EnhancementBiomedical ApplicationsHyperspectral Image Analysis

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.

Respond to this report

Responses (1)

Author Response

11 Feb 2025

Firman Alamsyah, Center for Medical Physics and Cancer Research, Ctech Labs Edwar Technology, Tangerang, 15143, Indonesia

We would like to thank the reviewer who have read our manuscript in detail and provided valuable input, as well as providing appropriate reference recommendations. We have made improvements to our manuscript based on the reviewers' suggestions and responded to all of the reviewers' notes, as we have written below.

1. We have revised the title to be shorter, more concise and informative, in order to attract readers' attention with clear and engaging language.

2. We do not know where to add “highlights bullets” and whether this is allowed or not by the journal editor. The importance of our research has been explained in the Introduction.

3. We have revised and re-edited the Abstract without eliminating any ideas to make it more consistent with the ideas presented in the manuscript and to make it more organized.

4. We have added details regarding sample size and the level of variability in observed results in the Abstract. We have replaced the word "safe" with another word that is more appropriate according to the context of the sentence.

5. We have omitted the repetition of points about the effects of electric fields to make it more concise. We have refined the hypothesis to be clearer and more specific. We have added detailed justification for the 100 kHz frequency compared to 150 kHz.

6. We have read the recommended studies provided by the reviewer and have compared them with the techniques and results of our study.

7. We only took 4 organs from 2 individual animals, and did not take organs from other individuals, because we considered the number of samples used for histological examination to be representative enough and had been approved by the ethics committee. We have added justification regarding the exposure duration of 10 hours per day for 21 days.

8. We have adequately contextualized the reduction in renal inflammation and hemorrhage with broader implications. We have adequately explained the relationship between fibrosis and chronic congestion. Based on suggestions from previous reviewers, we compared between 2 groups only, namely the effect of electric field on healthy rats (NIT vs NINT) and the effect of electric field on tumor-bearing rats (IT vs INT).

9. We have removed the term kidney safety and the sentence: "this therapy may be used to treat kidney injuries". We have added a clear rationale for using the frequency (100 kHz) and intensity (59-60 V/m) of ECCT in the Introduction section. We do not discuss these ECCT parameters in the Discussion section because these parameters are not the results of this study. We have added implications of using 100 kHz electric fields in non-cancer patients. We have combined several previous studies provided by the reviewer and compared them with our research techniques and results.

10. We have replaced all references published before 2011 with references published within the last 9 years. We also added several new references published in the last 10 years, including references recommended by the reviewer.

11. We've fixed typographical errors and broken up the overly long sentences into two shorter sentences.

View more View less

Competing Interests

No competing interests were disclosed.

Back to all reports

Reviewer Report

13 Views

26 Aug 2024 | for Version 4

Tejaswi Worlikar, University of Michigan, Ann Arbor, MI, USA

13 Views Cite this report Responses(1)

Approved With Reservations

The authors present a follow-up in vivo study investigating the safety of non-contact electric fields on healthy liver and kidney in Sprague Dawley breast cancer rat model using different settings of ECCT therapy compared to their original study. The following suggestions are provided to improve the quality of this manuscript:

1. Introduction:
"In our preliminary study using 9 mice, the intermediate-frequency (100 kHz) electric fields of Electro-Capacitive Cancer Therapy (ECCT) gave good results, wherein the tumour size was reduced by more than 67%, but showed no histological alterations in mammary and skin tissues."
Can you provide information on the effects of ECCT on tumor size in the current study? Did you observe tumor reduction? The previous published version of the study does not include any figures or tables showing the claimed 67% reduction in tumor size, can you please provide this as well?
2. Necropsy and organ harvesting: "Two kidneys and two livers from different rats were randomly collected from each group. A total of 16 organs were used for histological examination." If each group had 6 rats, can you please elaborate why only two kidneys and two rats were collected from each group? Is there no data available on the remaining 4 rats? If so, then the actual sample size for your safety analysis becomes quite small at 2 samples/group and 4 groups total.
3. Conclusions: "Exposure to this electric field can cause haemorrhagic damage in the livers of healthy rats, however, in other liver tissues and the kidneys, exposure to this electric field was safe. In addition, exposure to this electric field did not cause significant haemorrhagic damage in rats with breast cancer. It can even decrease the number of inflammations and haemorrhages in the kidneys." I am not sure if there is sufficient data presented to support the statement "can even decrease the number of inflammations and haemorrhages in the kidneys", since the sample size is low. A similar negative conclusion can be drawn saying "It can even increase the number of haemorrhages in the livers" (based on Table 4 data), but this would be inaccurate. Please consider rephrasing the conclusions.
4. Table 4 - Is this table showing the liver histopathological scoring results? Currently both Table 3 and Table 4 have the same heading. Please revise.
5. What are the differences in ECCT therapy parameters between the current study and the previous study "Nurhidayat L, Fajar I, Yati A, et al. 2022 [Ref-1]"? The current study uses 100 kHz and the previous study used 150 kHz, and both used 18 Vpp, is this assessment correct? Can you provide some insights into why these parameters were chosen/modified? Please mention the other study in your introduction, especially when it is claimed that this may be the first study to investigate the abnormalities in the kidney and liver under exposure to 100 kHz intermediate-frequency and low-intensity (50-60 V/m) non-contact electric fields.
6. "Damage to the kidneys and liver of the rats in the control group (NINT) cannot be predicted because rats with symptoms of illness had been excluded and rats were also randomly selected for each group." Were any rats excluded after the start of DMBA administration and/or ECCT therapy? If yes, what is the final size of each group? What types of illness were observed?
7. "Moreover, since this electric field exposure can reduce the number of inflammations and haemorrhages in the kidneys, this therapy can be used to treat kidney injuries or related diseases." Please consider rephrasing this claim.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

References

1. Nurhidayat L, Fajar I, Yati A, Prinanda H, et al.: Evaluation of Static Electric Field Exposure on Histopathological Structure and Function of Kidney and Liver in DMBA-Induced RAT (Rattus norvegicus Berkenhout, 1769). Malaysian Journal of Fundamental and Applied Sciences. 2022; 18 (6): 703-713 Publisher Full Text

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Small animal tumor models, cancer therapy, histotripsy

Respond to this report

Responses (1)

Author Response

22 Nov 2024

Firman Alamsyah, Center for Medical Physics and Cancer Research, Ctech Labs Edwar Technology, Tangerang, 15143, Indonesia

First of all we would like to apologize for the delay in responding to reviewer questions. We have answered the questions in the order given and have revised the article according to the reviewers' suggestions. We are very grateful to the reviewers who have evaluated this article and provided suggestions for improving this article. We hope that the reviewer will be willing to re-read the article that we have revised.

1. We realize that we did not include the graph of tumor size reduction of more than 67% in the previous publication that we cited in this article. However, we have data on tumor size reduction after therapy in publication number 9 (Figure 2) in the reference list: Alamsyah F, Pratiwi R, Firdausi N, et al.: Cytotoxic T Cells Response with Decreased CD4/CD8 Ratio During Mammary Tumors Inhibition in Rats Induced by Non-Contact Electric Fields. F1000 Res. 2021;10(35):1–21.

2. Yes, we only took samples of 2 kidneys and 2 livers from each group. We did not take kidneys and livers from all rats, so we do not have the data. The data of 2 kidneys and 2 livers from each group, we consider quite representative.

3. We have rephrased the conclusion as follows “Exposure to this electric field may cause haemorrhagic damage in the livers of healthy rats, however, in other liver tissues and the kidneys, exposure to this electric field was safe. In addition, exposure to this electric field did not cause significant haemorrhagic damage in rats with breast cancer and it may decrease the number of inflammations and haemorrhages in the kidneys of healthy rats.”

4. We have revised the heading of Table 4.

5. Yes, it is correct. We used a 100 kHz frequency electric field based on the results of a pilot study using mice exposed to a 100 kHz frequency electric field. We have also written in the Introduction that the electric field of 100 kHz gave the best results in our cell studies. Then we used a frequency of 150 kHz in a different study to investigate the difference in electric field frequencies in cancer therapy and also its safety on normal tissues. We have mentioned other study using 150 kHz frequency electric fields in the Introduction.

6. Rats were only excluded before and after the start of DMBA administration. We used 40 rats in total for this study and rats that had symptoms of illness were excluded before rats were assigned to each group. Rats that had symptoms of illness were rats that had lost weight. The final size of each group was 6 rats.

7. We have rephrased the sentence as follows “Moreover, since this electric field exposure may reduce the number of inflammations and haemorrhages in the kidneys, this therapy may be used to treat kidney injuries or related diseases.”

View more View less

Competing Interests

I do not have any competing interests.

Back to all reports

Reviewer Report

29 Views

22 Nov 2023 | for Version 3

Michael Staelens, Instituto de Física Corpuscular (IFIC), Paterna, Valencia, Spain

29 Views Cite this report Responses(1)

Not Approved

Although certain aspects of the article are improved, notable issues persist (several of which have already been mentioned yet remain unresolved), casting uncertainty over the study’s conclusions and hindering overall comprehension. In particular, numerous inconsistencies, insufficiencies in the presentation of the results, and a general lack of clarity pose significant challenges in following its content and undermine confidence in its conclusions. These issues must be resolved to ensure that the study and its conclusions can be accurately understood and interpreted by the scientific community. Specific issues that must be rectified—several of which have already been stated clearly in past reports—and various suggestions to facilitate timely revisions are as follows:

There appear to be serious inconsistencies between the conclusions of the study stated in various sections of the article.

The Abstract states, “The number of inflammation and hemorrhage in the kidney structure of the placebo group was the lowest and significantly different from the three other groups. All damages in the kidney were also found in the liver, but each showed different levels of damage. The damages in the kidney and liver caused by the exposure were not significant.”

Then, in the Discussion section, the following is stated: “In contrast to kidney histology, there was a significant damage, namely hemorrhage, in the liver of healthy rats (NIT group) after exposure to intermediate frequency non-contact electric fields”.

Further examples of these inconsistencies in the stated results/conclusions are present. This lack of alignment in reporting the findings of the study must be reconciled for clarity and to ensure an accurate interpretation of the study by the scientific community.
The presentation of results, in general, lacks clarity. It would be very helpful to the reader to include a table presenting all of the histopathological scoring results for the different groups (i.e., the mean ± SD for each of the groups for each of the parameters studied to quantify damage). This would enhance the clarity and transparency of the presented results and also allow readers to verify/reproduce the results of the statistical tests.
This is also the case regarding the results of the statistical hypothesis tests, in which the full results are not clearly presented in the manuscript. The inclusion of the results of these tests (possibly also in an additional table) would be beneficial in helping the reader to more easily identify and understand what differences were significant. Additionally, when significant differences, or the lack thereof, are presented, the p-value should be stated rather than simply p<0.05 or p>0.05; this is a better practice in scientific reporting and is important for the interpretation of the results so that the reader can understand not simply if a difference obtained in the study was significant or not, but also how significant (i.e., at what level of significance).
Many statements in the article regarding significant differences would benefit from stating the p-value for additional support and clarity around the significance of the observed difference. For example, “exposure to this non-contact electric field significantly decreased the number of inflammations and haemorrhages in the healthy rats (NIT group),” and “DMBA induction significantly damaged renal tubules.”
Some statements do not follow from the statistical test results reported and should be removed. For example, in the section on “Histopathology of liver”, the authors report “no significant difference in hemorrhage scores (p>0.05) in the livers of rats with breast cancer between the IT and the INT groups”; however, the next sentence reads, “Exposure to intermediate frequency electric field in the IT group slightly decreased hemorrhage in the liver compared to the INT group”. This directly contradicts the previous statement that no significant difference was found. Moreover, comparing the values reported, it is obvious to the reader that there is absolutely no statistically meaningful difference (0.87±0.56 vs. 0.88±0.46), i.e., no decrease that can or should be reported. The full passage is the following: “However, there was no significant difference in hemorrhage scores (p>0.05) in the livers of rats with breast cancer between the IT and the INT groups (Figure 5D). Exposure to intermediate frequency electric field in the IT group slightly decreased hemorrhage, cellular injury and congestion in the liver (0.87±0.56, 1.82±0.48, 0.37±0.56, respectively) after DMBA administration compared to the INT group (0.88±0.46, 1.96±0.51, 0.52±0.66, respectively).” Only the statistically meaningful differences should be reported as observed effects from the EF exposure and with the p-values explicitly stated.
Regarding the choice of statistical tests, the decision to analyze factors separately and exclusively conduct one-way tests is technically acceptable, albeit at the cost of sacrificing some information and statistical power. I suggest the authors consider one of the following options:

- Since the authors’ response states that they were unsuccessful in applying the SRH extension of the Kruskal–Wallis test, an alternative method that is highly applicable and more suitable for their study design than repeated one-way tests is ordinal logistic regression, which can be thought of as a multifactorial generalization of the Kruskal–Wallis test. This would ultimately be preferable to the statistical analysis methodology currently employed in the most recent version of the article and would enhance the interpretation of the results. For guidance on applying ordinal logistic regression in SPSS, please refer to the following page: https://statistics.laerd.com/spss-tutorials/ordinal-regression-using-spss-statistics.php.

- If the authors do not wish to attempt the previous suggestion or are unsuccessful in doing so, then the article should include at least some discussion on the rationale behind the chosen statistical testing methodology, as well as provide further insights into the drawbacks and caveats resulting from this choice.
The overall clarity and correctness of the writing still need work. Please perform a final series of revisions to the writing and unify English spellings. I strongly recommend using a free writing tool to help improve the writing (e.g., Grammarly), which would catch most of the remaining errors. A few examples:

- “In another study using electromagnetic field exposure to 150 kHz in healthy SD rats, showed normal kidney morphology, including normal-appearing glomeruli, tubules, and interstitium”. Revise to “Another study using electromagnetic field exposure at 150 kHz in healthy SD rats showed…”

- “The effects of non-contact electric fields exposure”. “electric fields exposure” -> ”electric field exposure”.

- “Fresh samples of the kidney and liver were collected for observing structural damage in both organs.” “for observing” -> “to observe”.

- Both “haemorrhage” and “hemorrhage” are written. Please maintain one choice of English spellings consistently and revise all instances of varied spellings to conform to this choice (preferably the former spelling, as the manuscript seems to largely conform to British English spellings).

- Number ranges, e.g., “50-60 V/m”, should use an en dash rather than a hyphen, i.e., 50–60 V/m.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Electromagnetic interactions with biological systems; non-invasive therapies; microtubules; biophysics; high energy physics; particle physics.

Respond to this report

Responses (1)

Author Response

11 Jan 2024

Firman Alamsyah, Center for Medical Physics and Cancer Research, Ctech Labs Edwar Technology, Tangerang, 15143, Indonesia

Dear reviewer,

I have revised the article and written my response according to your suggestions and requests.

1. There is an error in the article update carried out by F1000 Research. Specifically for the abstract, F1000 Research did not update revisions to the abstract in our article. The abstract written in the 2nd revision of our article is the first abstract we wrote. In addition, F1000 Research did not ask us for confirmation first, before the second revision of our article was published. We have reported the issue to the journal editor.

The original revision we wrote in the abstract was as follows: “All damages to the kidneys were also found in the liver, but each showed a different degree of damage. Exposure to this electric field can cause haemorrhagic damage to the livers of healthy rats, but not to rats with breast cancer.”. We have rewritten all revisions to the abstract.

2. Kidney and liver scoring and statistical analysis can be accessed on the link in the Underlying data section. However, we have presented the data in a table in our latest article revision according to the reviewer’s request.

3. Similar to response number 2, kidney and liver scoring and statistical analysis can be accessed on the link in the Underlying data section. However, we have added the p-values in our revised article according to the reviewer’s request in Table 3 and Table 4.

4. We have added the p-values in our revised article in Table 3 and Table 4.

5. We have removed the sentence that contradicts the statistical results.

6. We maintain the statistical tests used in our article and have added several reasons for determining these statistical tests along with their weaknesses.

7. We have used Grammarly to correct spelling mistakes in English in our revised article.

View more View less

Competing Interests

No competing interests were disclosed.

Back to all reports

Reviewer Report

26 Views

02 Jun 2023 | for Version 2

Michael Staelens, Instituto de Física Corpuscular (IFIC), Paterna, Valencia, Spain

26 Views Cite this report Responses(1)

Not Approved

The revised version of the manuscript, “Kidney and liver histology in tumour-induced rats exposed to non-contact electric fields”, by Alamsyah et al., is an improvement over the first version, with many of the reviewers’ suggestions implemented. However, Major Concern #1 from my first review of the manuscript does not appear to have been taken into account; despite the authors’ claim that a new statistical analysis was performed, the Data Analysis section still states that “scoring results were then analysed statistically to determine significant differences among groups (p<0.05) using the Kruskal-Wallis test followed by the Mann-Whitney test”. As highlighted in my first review of the manuscript, this is an incorrect choice of statistical analysis methodology. This is problematic regarding both the interpretation of the data and the subsequent conclusions drawn from the study. Two alternative non-parametric methods (i.e., do not require normally distributed data) that can be used when a measure is affected by two (or more factors) and are thus applicable to the results reported by the authors in the manuscript were suggested; however, neither one appears to have been implemented in the revisions.

The alternating “non-contact electric fields” therapy studied by the authors is nearly identical to the relatively recently FDA-approved alternating electric field therapy—often referred to in the literature as tumor-treating fields—which (instead) typically uses electrodes placed on the skin and has shown remarkable efficacy as a non-invasive anticancer treatment modality. Consequently, the most important aspects of the study reported by the authors (as far as I can tell) are how the experimental group that received both tumor induction and EF therapy compares to the other experimental/treatment groups and the control group, as well as how the EF-only group and the control group compare (which is interesting for establishing effects of the treatment on healthy/non-tumoral cells). There are four groups reported in the manuscript, control (NINT), EF only (NIT), tumor induction only (INT), and the combined tumor induction and EF therapy group (IT). The groups thus differ very clearly by two independent variables (comprising two levels each): EF treatment (yes/no) and tumor induction (yes/no). The Kruskal–Wallis test cannot be used to compare these groups; the same is true of the Mann–Whitney test, which assumes that the groups differ by only one independent variable. To phrase it differently, the Kruskal–Wallis test is a one-way test; comparing the control to the IT group, for example, involves comparing groups that differ in two ways. A simultaneous comparison between the IT group and the three other groups must be performed in order to correctly establish the influence of the two different independent variables on the dependent variable measured (i.e., the histopathological scoring results) and to determine the main effect of each independent variable as well as to determine if there is any interaction between the two independent variables.

“[The] selection of [an] appropriate statistical method is [a] very important step in [the] analysis of biomedical data. A wrong selection of the statistical method not only creates some serious problem[s] during the interpretation of the findings but also affects the conclusion[(s)] of the study”¹. Consequently, the key results and conclusions stated in the manuscript might be erroneous (i.e., false claims of significance/insignificance). Thus, the status of my review of the manuscript remains “not approved”. The problem is that without correctly applying an appropriate statistical analysis method that suits the data collected and the results obtained, the actual findings and conclusions of the study are unclear. Therefore, until this major concern is resolved, I do not believe that the manuscript and the reported results should be indexed or cited in order to prevent the propagation of potentially misleading results and conclusions.

References

1. Mishra P, Pandey CM, Singh U, Keshri A, et al.: Selection of appropriate statistical methods for data analysis.Ann Card Anaesth. 2019; 22 (3): 297-301 PubMed Abstract | Publisher Full Text

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Electromagnetic interactions with biological systems; non-invasive therapies; microtubules; biophysics; high energy physics; particle physics.

Respond to this report

Responses (1)

Author Response

07 Nov 2023

Firman Alamsyah, Center for Medical Physics and Cancer Research, Ctech Labs Edwar Technology, Tangerang, 15143, Indonesia

Dear Reviewer,

We had difficulty carrying out statistical tests with 2 factors using the suggested test. We have tried it but it did not work. We had difficulty finding examples of the use of the test in published articles. We have also asked biostatisticians for help, but none of them can do it, including one of our colleagues who is the head of the Biostatistics Department at a university.

Therefore, we carried out repeated statistical tests using only 1 factor, namely exposure to electric fields, by comparing the level of damage to kidney and liver tissues. We got different results from previous statistical tests. Based on the statistical results, we have replaced the graphs in Figures 3 and 5, and revised the Abstract, Results, and Discussion sections.

View more View less

Competing Interests

No competing interests were disclosed.

Back to all reports

Reviewer Report

71 Views

02 Mar 2023 | for Version 1

Michael Staelens, Instituto de Física Corpuscular (IFIC), Paterna, Valencia, Spain

71 Views Cite this report Responses(1)

Not Approved

There are many problems with the writing regarding grammar, punctuation, missing spaces, article usage problems, misspelled words, incorrect verb forms, faulty tense sequences, incomplete sentences, and several other sentences that simply do not make sense. In aggregate, these errors severely hinder the readability and clarity of the manuscript. I strongly recommend that the authors consider sending the manuscript to a professional English editing service, or at the very least, use one of the numerous free grammar and writing tools available online to assist with correcting many of the writing issues.
The manuscript mixes both U.S. and U.K. spellings, e.g., “tumor” and “tumour” are both used, as are “acclimatization” and “randomisation” (z vs. s). Please maintain one choice consistently in your writing.
The manuscript lacks coherence and does not communicate a cohesive story. Here are some examples of inconsistencies that negatively affect the cohesion:

- In the “Results” part of the Abstract, it reads, “The damages in the kidney and liver caused by the exposure were not significant.”, but then in the Discussion section, the following is stated “Since the significant damages of the glomerulus were observed in the kidneys of the placebo (NIT)…”, followed by, “both DMBA administration and exposure to non-contact electric fields affected the thickening of Bowman’s capsule”, which certainly sounds like there were significant damages observed in the kidney samples taken from the group that only received exposure to the electric fields (NIT).

- The need for studying the effects of electric fields-based cancer therapies on healthy tissues was motivated in the Introduction by pointing out that there might be interactions between the kidney/liver and electrostatic waves; however, the electric fields-based cancer therapies referenced and discussed in the manuscript (and that are actually used in a clinical setting) do not employ static fields, but rather alternating electric fields.

- Several of the references cited in the Discussion section that are discussed with regards to the interpretation of the results studied vastly different parameters for the electric fields than those employed in the experiments reported in the manuscript and that are typically used to treat cancers with alternating electric field therapy (e.g., 575 V/cm in one of the studies cited vs. the low-intensity of only a few V/cm maximum for alternating electric field therapy).
The manuscript often mentions the significance of results/effects; however, none of these statements include the supporting quantitative results that suggest the differences obtained are, in fact, significant (or not). Consequently, the level of significance associated with these statements is also not clear to the reader (without reading through the data files provided alongside the manuscript). Please revise all these statements to include the quantitative results of the statistical analyses that support the stated significances. For example, the following sentence on page 5, “The main damage found in the kidney glomerular was the thickening of the Bowman capsule whose scores were significant in all treatment groups (1.12±0.56 for NIT, 1.16±0.74 for INT, and 1.24±0.59 for IT groups) compared to the control (NINT) group (0.88±0.56).”, should be revised to include the corresponding H-values (or p-values, etc.) that supports the statement that these differences between scores were significant (“in all treatment groups compared to the control group”). This makes it both clear to the reader that the differences between groups are indeed significant based on the statistical analysis results obtained and provides the reader with the quantitative results, so they also get a sense of how significant (or insignificant) each particular result is without having to dig through spreadsheets of data while reading the article.
The quality of the plots provided in Figures 2 and 4 is quite low. Additionally, the dots used to represent the data are very small and difficult to distinguish (when reading the article on a tablet at 100% magnification). Please consider reproducing these plots at a higher quality (600 DPI or greater) and with the points used to represent the data enlarged and spread out more clearly, so the individual points are distinguishable without needing to zoom in (which also leads to substantial blurring due to the low quality of the plots).

Specific Comments:
Major concerns:

The data analysis section states that the scoring results were analyzed statistically using the Kruskal–Wallis test; however, the scoring results were obtained for four different groups that differed independently in two ways (i.e., there were two independent variables involved—tumour induction (or not) and treatment (or not)). Consequently, the Kruskal–Wallis test, which is only applicable in the one-factor case, cannot be used. Due to this problem, the actual statistical significance of the obtained differences between the various groups studied is not clear, and the soundness of the conclusions drawn is uncertain. If you do require a non-parametric rank-based method, then consider the Scheirer–Ray–Hare extension of the Kruskal–Wallis test, which can be used in the case where a measure may be affected by two or more factors. Alternately, the (two-factor) aligned rank transform analysis of variance (ART-ANOVA) may also be suitable.
Moreover, the conclusions do not appear to be clearly supported by the results. This concern is not only due to the previous major comment noted but also due to the following:

- The first conclusion drawn is that “The non-contact electric fields were not harmful to the renal and liver structure of tumour-induced rats"; however, one of the outcomes described in the Results and Discussion sections was that the non-contact low-intensity electric fields significantly "affected the thickening of Bowman's capsule" (NINT versus NIT), which is apparently indicative of renal damage. Consequently, I am not sure that this general conclusion is completely supported by the results of this study.

- The second conclusion drawn is that non-contact electric fields “may optimise/increase the renal function in normal rats”. What results obtained in this study demonstrate an optimization and/or increase in renal function in normal rats exposed to low-intensity non-contact electric fields? The only discussion in the manuscript regarding renal function appears to be that provided on page 8; however, the discussion there mentions renal function impairment as a possible result of DMBA-induced inflammation and does not discuss electric fields. At the end of this paragraph, non-contact electric fields are mentioned: “non-contact electric fields decreased the number of inflammations and haemorrhages in the placebo (NIT) group, as shown in Figure 2”; however, the statistical significance of this difference is not stated. Is the decrease being mentioned here actually meaningful statistically? And if so, how statistically significant was this decrease, and how exactly does this marginal decrease in observed inflammations and haemorrhages connect to the conclusion that renal function has been optimized due to treatment with non-contact electric fields? As far as I can tell, no tests or metrics that could be used to accurately evaluate any changes in kidney and/or liver function and that would support this particular conclusion were included in the experiments and analyses reported in the manuscript.
Why were damages also exhibited in the control (NINT) group? Naively, I would not have expected this outcome, and it is not explained or discussed in the manuscript.

Due to these major concerns, I do not believe that the conclusions stated in the manuscript are clearly supported by the results.

Minor concerns:

More details regarding both the electric field used in the experiments and how it was applied should be provided for clarity and reproducibility of the study:

- How were the electric fields generated? What device was used (make, model, etc.)?

- Were the electric fields static or alternating?

- What was the duty cycle?

- Given that the rats were exposed to non-contact electric fields, I assume that the electrodes were not fixed to the skin of the rats and that they received whole-body exposure to the electric fields. Is this true? Additionally, how many electrodes were used? where were they placed/attached? and with what orientation? In other words, how many electric fields were there, were they uniform or non-uniform, and with what directionality(ies)?

Please provide this information in the manuscript.
While the exposures were being performed, were there any other sources of electromagnetic radiation in the room that could be considered a source of interference?
On page 3, the last sentence in the Introduction states, “According to our knowledge, this is the first study investigating the abnormalities in the kidney and liver under exposure to 100 kHz intermediate frequency and low-intensity non-contact electric fields”, which does appear to technically be true; however, a very similar¹ study was recently published that reports the results of analyzing histological profiles of vital organs of Sprague Dawley rats exposed to 150 kHz low-intensity non-contact electromagnetic radiation. It would be valuable and interesting to revise the discussion in the manuscript to compare and contrast your results and conclusions obtained versus those reported in this very similar study in the literature.
On page 3, a value of 18 V_pp is noted after stating that the electric fields used in the study are low intensity. The electric field intensity has units of electric potential per distance (V/m in SI units), not simply volts, which is the unit of electric potential. Please revise accordingly (including at the bottom of page 3, where the same issue occurs again).
On page 3, it is stated that “Although non-contact electric fields-based therapy has the potential to treat cancer, the safety of this kind of therapy when treating healthy tissues should be investigated. This is because injuries may occur after exposure to electric fields due to the dielectric property of the kidney and liver, which may interact with electrostatic waves. Therefore, it is important to investigate the abnormalities in the kidney and liver under exposure to electric fields during cancer treatment.” The second sentence quoted here does not connect to the previous or subsequent sentences; the electric fields used in cancer treatment that this article alludes to, so-called TTFields, are alternating electric fields (time-varying), whereas electrostatic always refers to time-invariant electric fields. Please revise.
Additionally, on page 3, the sentence “the proliferation of cancer cells was successfully inhibited under exposure to intermediate frequency and low-intensity electric fields” should be revised to read “low-intensity alternating electric fields”. This statement would also benefit from more specificity, i.e., what types of cancer cells was this inhibition demonstrated for in the studies cited here and with what parameters for the electric fields employed (frequency, intensity, duration of exposure)?
Should the y-axis label for Figure 2d read “Congestion score” instead? (To maintain consistency with the other subfigures presented in Figure 2 and with Figure 4c.)
In Figure 4, the values of mean ± SD noted in each of the subfigures use inconsistent numbers of significant digits. Specifically, the values of the uncertainties are reported with greater precision than the mean values themselves, which indicates that the errors are known more precisely than the values and is very unusual.
Figures 2 and 4 include a footnote partially describing the meaning of the labels “a”, “b”, etc. as indicating significance; however, the difference between each of these labels is not clear. The exact meaning of “a”, “b”, etc. should be clarified in the notes provided under Figures 2 and 4. Additionally, while it is clear that, for example, in Figure 2a, the NINT group has the label “a” to denote that it is significant, it is not clear what it is being compared to, i.e., significant compared to which group(s)? Lastly, please change “different words a, b, c” to read “different letters” or “different labels”.
In the second paragraph of the Discussion on page 7, it is mentioned that “some biological effects of exposure to electric fields (0.6 and 340 kV/m) were revealed in humans and vertebrates, but no histological abnormalities were found in the organs, including the kidneys.” Comparing with this study does not make much sense for the following reasons:

- The particular study cited here evaluated the biological effects of static electric fields, which are not the same as the alternating electric fields exploited in cancer therapy, and that the manuscript purportedly studies the effects of.

- The parameters stated (0.6 and 340 kV/m) corresponding to this particular study that is being referenced are NOT consistent with the low-intensity electric fields used in cancer therapy (approx. 1–3 V/cm) and, again, that the manuscript appears to be studying.
The abbreviation ECCT is not defined anywhere in the manuscript.
The first sentence in the last paragraph of the Discussion section (page 9) does not make sense. Specifically, what does “Based on the evidence for the efficacy and safety of normal tissues and organs...” mean? I believe that rather than describing evidence on the “efficacy and safety of a normal tissue”, what is trying to be said here is something like, “Based on the evidence for the efficacy and safety of non-contact low-intensity electric fields regarding normal tissues and organs...”, or something along these lines. Please rephrase this sentence accordingly.
The statement regarding the role of the funders, “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”, should also mention the role of the funders regarding the interpretation of the data/results.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

No

References

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Electromagnetic interactions with biological systems; microtubules; biophysics; high energy physics; particle physics.

Respond to this report

Responses (1)

Author Response

23 Mar 2023

Firman Alamsyah, Center for Medical Physics and Cancer Research, Ctech Labs Edwar Technology, Tangerang, 15143, Indonesia

We thank you for all the comments and suggestions and we will revise our paper. Below are our answers to the comments and suggestions provided in order.

General comments
1. We have used a professional English editing service (PaperTrue) before submitting this article. We will do one more proofreading.

2. We will use UK spelling in our revised article.

3. We will revise the abstract so that it is coherent with the contents of the paper. We will replace some of the references in the Discussion section. Previously, we did not find any references in accordance with the study conducted.

4. In the Data Analysis section and in the Figures, we have mentioned a p<0.05 value for a significant difference among groups. We will rewrite it for each data in the Results section.

5. We will fix Figures 2 and 4.

Specific comments
Major concerns
1. We will evaluate the statistical tests performed.

2. We will revise the Conclusions section to suit the Results and Discussion sections.

3. Damage to the kidneys and liver of the rats in the control group (NINT) cannot be predicted because we have excluded rats with symptoms of illness as stated in the article. Rats were also randomly selected for each group as stated also in article.

For damage to the kidney in the NINT group, if we look at the interstitial tissue, a score below 2 indicates that there is little inflammation or hemorrhage. Inflammation is part of the activation of the immune system in response to acute or chronic kidney injury which can be caused by pathogens that enter the rat’s body (Imig & Ryan, 2013).

For damage to the liver in the NINT group, if we look at the hemorrhagic and congestion scores which are below 1, this indicates that there is little or no damage to the liver. For a cellular damage score below 2, this indicates reversible damage with less than 15% necrosis. Liver hepatocytes have many vital functions, so they can proliferate extensively, which allows efficient regeneration of the liver for reversible damage (Chen et al., 2020). In addition, the liver itself is a very vulnerable organ due to its size and is the organ most frequently injured after abdominal trauma (Bilgic et al., 2014).

Minor concerns
1. We will provide more detailed information about the electric fields used in the experiment in our revised article.

2. The experiment was carried out in a special room which only contained experimental animal cages.

3. We will revise the Discussion section using the appropriate references.

4. We will include electric fields intensity data in our revised article.

5. We will revise this section (page 3).

6. We will revise this section (page 3).

7. We will revise Figure 2d.

8. We will revise Figure 4.

9. We will revise Figures 2 and 4.

10. We will revise the Discussion section using the appropriate references.

11. We will write what ECCT stands for in our revised article.

12. We will rephrase this sentence. There may be phrases lost in the editing process.

13. We will add the statement regarding the role of the funders in the interpretation of the data/results.

References
Bilgiç I, Gelecek S, Akgün AE, et al.: Evaluation of liver injury in a tertiary hospital: a retrospective study. Ulus Travma Acil Cerrahi Derg. 2014; 20(5): 359-365.

Chen F, Jimenez RJ, Sharma K, et al.: Broad Distribution of Hepatocyte Proliferation in Liver Homeostasis and Regeneration. Cell Stem Cell. 2020; 26(1):27-33

Imig JD, Ryan MJ: Immune and Inflammatory Role in Renal Disease. Compr. Physiol. 2013; 3(2): 957-976.

View more View less

Competing Interests

No competing interests were disclosed.

Back to all reports

Reviewer Report

53 Views

27 Feb 2023 | for Version 1

Chandran Nadarajan, School of Medical Sciences, Hospital Universiti Sains Malaysia, Kota Bharu, Malaysia

53 Views Cite this report Responses(2)

Approved

Why do the authors choose the voltage 100kHz and 18v, specifically? Were the preliminary results referred to published? Are there any other articles supporting the usage of this voltage and frequency?
Is there any statistical analysis done? This will help strengthen the conclusion.
Why was there damage in the kidney interstitial tissue and liver damage in the NINT group?

Figures 1 and 3 are very small, with many icons. Please enhance them.
Figures 2, and 4 suggest changing to the Whisker box plot and being made bigger. It will be better.
Utilization of rank order, like Mann Whitney, might help to bring forward the idea better.
Conclusion is a little too short and needs to be improved. Adding limitations and further direction might help. This can be done in the discussion.
IRB date, number, and place should be shown.
In several places, language is unclear, ambiguous, or confusing. It is necessary to use a professional editing service to improve it before indexing.
I think the title "Effects of non-contact electric fields on kidney and liver histology in tumour-induced rats." Might be more appropriate.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

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

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Interventional oncology, interventional and diagnostic radiology

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.

Respond to this report

Responses (2)

Author Response

01 Mar 2023

Firman Alamsyah, Center for Medical Physics and Cancer Research, Ctech Labs Edwar Technology, Tangerang, 15143, Indonesia

I will answer the reviewer's comments sequentially in the order of the comments.

1. We have conducted an in vitro study using various frequencies and intensities, but this study has not been published. We used 100 kHz and 18 Vpp for in vivo study because this frequency and intensity gave the best results in in vitro study, where 28-39% of breast cancer cells died (Alamsyah et al., 2015). In the preliminary in vivo study using 9 mice, the frequency of 100 kHz and intensity of 18 Vpp also gave good results, where the tumor size was reduced by more than 67% and the results of histopathological analysis on normal skin and breast tissue, showed no damage (Alamsyah et al., 2015).

2. Yes, we did the statistical analysis and it has been written in the article in the Data Analysis section. Kidney and liver scoring data, as well as statistical analysis can be seen in the links provided in the Data Availability section of the article.

3. Damage to the kidneys and liver of the rats in the control group (NINT) cannot be predicted, because we have excluded rats with symptoms of illness as stated in the article. Rats were also randomly selected for each group as stated also in article.

For damage in the renal interstitial tissue of the NINT group, a score below 2 indicated that there was little inflammation or hemorrhage. Inflammation is part of the activation of the immune system in response to acute or chronic kidney injury which can be caused by pathogens that enter the rat’s body (Imig & Ryan, 2013).

For damage in the liver of the NINT group, if we look at the hemorrhagic and congestion scores which are below 1, this indicated that there was little or no damage to the liver. For a cellular damage score below 2, this indicated reversible damage with less than 15% necrosis. Liver hepatocytes have many vital functions, so they can proliferate extensively, which allows efficient regeneration of the liver for reversible damage (Chen et al., 2020). In addition, the liver itself is a very vulnerable organ due to its size and is the organ most frequently injured after abdominal trauma (Bilgic et al., 2014). Rats are active animals and may chase or fight each other in communal cages which can cause trauma to their body (Steimer, 2011).

4. We found no reference to kidney or liver damage at intermediate frequency and low intensity electric fields, especially 100 kHz and 18 Vpp. We have also stated this in the article. In the Introduction section, we have also stated that this is the first study to investigate abnormalities in the kidney and liver under exposure to a intermediate frequency of 100 kHz and a low intensity non-contact electric field.

We will add necessary information to our article from the answers to the reviewer's questions.

Thank you.

References
Alamsyah F, Ajrina IN, Dewi FN, et al.: Antiproliferative Effect of Electric Fields on Breast Tumor Cells In Vitro and In Vivo. Indones. J. Cancer Chemoprev. 2015; 6(3): 71–77.
Bilgiç I, Gelecek S, Akgün AE, et al.: Evaluation of liver injury in a tertiary hospital: a retrospective study. Ulus Travma Acil Cerrahi Derg. 2014; 20(5): 359-365.
Chen F, Jimenez RJ, Sharma K, et al.: Broad Distribution of Hepatocyte Proliferation in Liver Homeostasis and Regeneration. Cell Stem Cell. 2020; 26(1):27-33
Imig JD, Ryan MJ: Immune and Inflammatory Role in Renal Disease. Compr. Physiol. 2013; 3(2): 957-976.
Steimer T: Animal models of anxiety disorders in rats and mice: some conceptual issues. Trans Res. 2011; 495-506.

View more View less

Competing Interests

No competing interests were disclosed.

Author Response

21 Mar 2023

Firman Alamsyah, Center for Medical Physics and Cancer Research, Ctech Labs Edwar Technology, Tangerang, 15143, Indonesia

We thank you for the additional suggestions and we will revise our paper. Below are our answers to the suggestions provided in order.

1. We will enhance Figure 1 and Figure 3 in our revision.

2. We will consider to use the Whisker box plot in Figure 2 and Figure 4.

3. We have conducted Mann-Whitney test (α=0.05) in our statistical analysis.

4. We will improve the conclusions of our study and we will discuss the limitations of this study. We have written further directions of this study at the end of the discussion section.

5. IRB date, number, and place have been written in the ethical approval section.

6. We have used a professional editing service (PaperTrue) before submitting this article. We will do one more proofreading.

7. We will improve the title of this article.

View more View less

Competing Interests

No competing interests were disclosed.

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

[1] 1. Zhang Y, Ding J, Duan W, et al.: Influence of Pulsed Electromagnetic Field with Different Pulse Duty Cycles on Neurite Outgrowth in PC12 Rat Pheochromocytoma Cell. Bioelectromagn. 2005; 26: 406–411. PubMed Abstract | Publisher Full Text

[2] 2. Qiao G, Duan W, Chatwin C, et al.: Electrical Properties of Breast Cancer Cells from Impedance Measurement of Cell Suspensions. J. Phys. Conf. Ser. 2010; 224: 1–4.

[3] 3. Kirson ED, Dbaly V, Tovarys F, et al.: Alternating Electric Fields Arrest Cell Proliferation in Animal Tumor Models and Human Brain Tumors. PNAS. 2007; 104(24): 10152–10157. PubMed Abstract | Publisher Full Text | Free Full Text

[4] 4. Fu F, Xin SX, Chen W: Temperature- and Frequency-Dependent Dielectric Properties of Biological Tissues within the Temperature and Frequency Ranges Typically Used for Magnetic Resonance Imaging-Guided Focused Ultrasound Surgery. Int. J. Hyperth. 2014; 30(1): 56–65. PubMed Abstract | Publisher Full Text

[5] 5. Ji Z: Expanded Modeling of Temperature-Dependent Dielectric Properties For Microwave Thermal Ablation. Phys. Med. Biol. 2011; 56(16): 5249–5264. PubMed Abstract | Publisher Full Text | Free Full Text

[6] 6. Pop M, Molckovsky A, Chin L, et al.: Changes in Dielectric Properties at 460 kHz of Kidney and Fat During Heating: Importance for Radio-Frequency Thermal Therapy. Phys. Med. Biol. 2003; 48: 2509–2525. PubMed Abstract | Publisher Full Text

[7] 7. Porter E, Gioia AL, Santorelli A, et al.: Modeling of the Dielectric Properties of Biological Tissues within the Histology Region. IEEE Trans. Dielectr. Electr. Insul. 2017; 24(5): 3290–3301. Publisher Full Text

[8] 8. Alamsyah F, Ajrina IN, Dewi FN, et al.: Antiproliferative Effect of Electric Fields on Breast Tumor Cells In Vitro and In Vivo. Indones. J. Cancer Chemoprev. 2015; 6(3): 71–77.

[9] 9. Alamsyah F, Pratiwi R, Firdausi N, et al.: Cytotoxic T Cells Response with Decreased CD4/CD8 Ratio During Mammary Tumors Inhibition in Rats Induced by Non-Contact Electric Fields. F1000 Res. 2021; 10(35): 1–21.

[10] 10. Mujib SA, Alamsyah F, Taruno WP: Cell Death and Induced p53 Expression in Oral Cancer, HeLa, and Bone Marrow Mesenchyme Cells Under the Exposure to Noncontact Electric Fields. Integ. Med. Int. 2017; 4: 161–170. Publisher Full Text

[11] 11. Pratiwi R, Antara NY, Fadliansyah LG, et al.: CCL2 and IL18 Expressions May Associate with the Anti-Proliferative Effect of Noncontact Electro Capacitive Cancer Therapy In Vivo. F1000 Res. 2019; 8(1770): 1–14.

[12] 12. Yang M, Brackenbury WJ: Membrane Potential and Cancer Progression. Front. Physiol. 2013; 4(185): 1–10.

[13] 13. Kadir LA, Stacey M, Jolley RB: Emerging Roles of the Membrane Potential: Action Beyond the Action Potential. Front. Physiol. 2018; 9(1661): 1–10.

[14] 14. Bray F, Ferlay J, Soerjomataram I, et al.: Global Cancer Statistics 2018: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J. Clin. 2018; 68: 394–424. PubMed Abstract | Publisher Full Text

[15] 15. Costa E, Ferreira-Gonçalves T, Chasqueira G, et al.: Experimental Models as Refined Translational Tools for Breast Cancer Research. Sci. Pharm. 2020; 88(32): 1–29.

[16] 16. Abbasalipourkabir R, Dehghan A, Salehzadeh A, et al.: Induction of mammary gland tumor in female Sprague Dawley rats with LA7 cells. Afr. J. Biotechnol. 2010; 9(28): 4491–4498.

[17] 17. Akla B, Monteil J, Paraf F, et al.: A New Orthotopic Model of Human Breast Cancer in Immunocompetent Rats. Anticancer Res. 2003; 23(5A): 3761–3766. PubMed Abstract

[18] 18. Bancroft JD, Cook HC: Manual of Histology Techniques and Their Diagnostic Application. London:Churchill Livingstone;1994.

[19] 19. Gibson-Corley KN, Olivier AK, Meyerholz DK: Principles for Valid Histopathologic Scoring in Research. Vet. Pathol. 2013; 50(6): 1–22.

[20] 20. Khalid U, Pino-Chavez G, Nesargikar P, et al.: Kidney Ischaemia Reperfusion Injury in the Rat: The EGTI Scoring System as a Valid and Reliable Tool for Histological Assessment. J. Histo. Histopathol. 2016; 3(1): 1–7. Publisher Full Text

[21] 21. El-Gerbed MSA: Silymarin, Protects Against 7,12Dimethyl-benz [A] anthracene–Induced Hepatotoxicity in Albino Rats. RJPBCS. 2013; 4(3): 1534–1548.

[22] 22. Elbaz A, Ghonimi WAM: Exposure Effects of 50 Hz, 1 Gauss Magnetic Field on the Histoarchitecture Changes of Liver, Testis and Kidney of Mature Male Albino Rats. J. Cytol. Histol. 2015; 06(4): 1–6. Publisher Full Text

[23] 23. Duarte R, Jesus P, Farinha R, et al.: Adipose Tissue and Liver in DMBA Experimental Intoxication. Exp. Pathol. Health Sci. 2016; 8(1): 59:66.

[24] 24. Kotyk T, Dey N, Ashour AS, et al.: Measurement of the Glomerulus Diameter and Bowman’s Space Thickness of Renal Albino Rats. Comput. Methods Prog. Biomed. 2016; 126: 143–153. PubMed Abstract | Publisher Full Text

[25] 25. Paliwal R, Sharma V, Pracheta, et al.: Anti-Nephrotoxic Effect of Administration of Moringa oleifera Lam in Amelioration of DMBA-Induced Renal Carcinogenesis in Swiss Albino Mice. Biol. Med. 2011; 3(2): 27–35.

[26] 26. Lee JS, Ahn SS, Jung KC, et al.: Effects of 60 Hz Electromagnetic Field Exposure on Testicular Germ Cell Apoptosis in Mice. Asian J. Androl. 2004; 6(1): 29–34. PubMed Abstract

[27] 27. Sharma V, Paliwal R: Chemo Protective Role of Moringa oleifera and Its Isolated Saponin Against DMBA Induced Tissue Damage in Male Mice: A Histopathological Analysis. Int. J. Drug Dev. Res. 2012; 4(4): 215–228.

[28] 28. Rems L, Kasimova MA, Testa I, et al.: Pulsed Electric Fields Can Create Pores in the Voltage Sensors of Voltage-Gated Ion Channels. Biophys. J. 2020; 119: 190–205. PubMed Abstract | Publisher Full Text | Free Full Text

[29] 29. Petri AK, Schmiedchen K, Stunder D, et al.: Biological Effects of Exposure to Static Electric Fields in Humans and Vertebrates: A Systematic Review. Environ. Health. 2017; 16(41): 1–23.

[30] 30. Sharma V, Paliwal R, Janmeda P, et al.: Renoprotective Effects of Moringa oleifera Pods in 7,12-Dimethylbenz [a]anthracene-Exposed Mice. J. Chin. Integr. Med. 2012; 10(10): 1171–1178. Publisher Full Text

[31] 31. Neal RE, Garcia PA, Kavnoudias H, et al.: In Vivo Irreversible Electroporation Kidney Ablation: Experimentally Correlated Numerical Models. IEEE Trans. Biomed. Eng. 2015; 62(2): 561–569. Publisher Full Text

[32] 32. Cachofeiro V, Goicochea M, Vinuesa SG, et al.: Oxidative Stress and Inflammation, A Link Between Chronic Kidney Disease and Cardiovascular Disease. Kidney Int. 2008; 74: S4–S9. Publisher Full Text

[33] 33. Li N, Karin M: Is NF-kappaB the Sensor of Oxidative Stress? FASEB J. 1999; 13(10): 1137–1143. Publisher Full Text

[34] 34. Kandeel M, Abdelaziz I, Elhabashy N, et al.: Nephrotoxicity and Oxidative Stress of Single Large Dose or Two Divided Doses of Gentamicin in Rats. Pak. J. Biol. Sci. 2011; 14: 627–633. Publisher Full Text

[35] 35. Kesba HH, El-Belagi HS: Biochemical Changes in Grape Rootstocks Resulted from Humic Acid Treatments in Relation to Nematode Infection. Asian Pac. J. Trop. Biomed. 2012; 2(4): 287–293. Publisher Full Text

[36] 36. Bharali R, Tabassum J, Azad MRH: Chemomodulatory Effect of Moringa oleifera, Lam, on Hepatic Carcinogen Metabolising Enzymes, Antioxidant Parameters and Skin Papillomagenesis in Mice. Asian Pac. J. Cancer Prev. 2003; 4(2): 131–139. PubMed Abstract

[37] 37. Koul A, Arora N, Tanwar L: Lycopene Mediated Modulation of 7,12 Dimethlybenz(A) anthracene Induced Hepatic Clastogenicity in Male Balb/c Mice. Nutr. Hosp. 2010; 25(2): 304–310. PubMed Abstract

[38] 38. de Oliveira KD , Avanzo GU, Tedardi MV, et al.: Chemical Carcinogenesis by DMBA (7,12-Dimethylbenzanthracene) in Female BALB/c Mice: New Facts. Braz. J. Vet. Res. Anim. Sci. 2015; 52(2): 125–133. Publisher Full Text

[39] 39. Gutierrez G, Reines HD, Wulf-Gutierrez ME: Clinical Review: Hemorrhagic Shock. Crit. Care. 2004; 8: 373–381. PubMed Abstract | Publisher Full Text | Free Full Text

[40] 40. Chen W, Wang L, Liang P, et al.: Reducing ischemic kidney injury through application of a synchronization modulation electric field to maintain Na⁺/K⁺-ATPase functions. Sci. Transl. Med. 2022; 14(635): eabj4906. Publisher Full Text

[41] 41. Alamsyah F: Kidney and Liver Histology in Tumour-induced Rats Exposed to Non-contact Electric Fields. [Dataset]. OSF. 2022 October 22. Publisher Full Text

Kidney and liver histology in tumour-induced rats exposed to non-contact electric fields

Abstract

Keywords

Introduction

Methods

Experimental design

Necropsy and organ harvesting

Renal histopathological analysis

Table 1. Histopathological scoring system for the kidney.

Liver histopathological analysis

Table 2. Histopathological scoring system for the liver.

Data analysis

Results

Histopathology of kidney

Figure 1. Histological features of tubular, interstitial, glomerular, and congestion damages in rat renal sections with H&E staining.

Figure 2. Scoring of tubular, interstitial, glomerular, and congestion damages in rat renal sections.

Histopathology of liver

Figure 3. Histological features of haemorrhage, congestion, and cellular damages in rat liver section with H&E staining.

Figure 4. Scoring of cellular damages, haemorrhage, and congestion in rat liver sections.

Discussion

Conclusions

Ethical approval

Data availability

Underlying data

Extended data

Reporting guidelines

Acknowledgements

References

Comments on this article Comments (0)

Open Peer Review

Comments on this article Comments (0)

Open Peer Review

Reviewer Status

Reviewer Reports

Comments on this article

Browse by related subjects

Competing Interests Policy

Stay Updated