Effects of non-contact electric fields on kidney and liver histology in tumour-induced rats

Introduction

The knowledge that electric fields can induce biological effects came to light in the 19^th century. Many studies have been conducted which provide evidence that exposure to electric fields can produce alterations in living things.¹ Several studies have examined the effects produced by electric fields on cell function.² Kirson et al.³ reported that electric field intensity inside the cell is less than 10 V/cm, but inside the cell membrane, it can reach 10⁵ V/cm. At the organ level, the kidney and liver have dielectric properties that exhibit a time-temperature dependence.^4–6 Therefore, they possess both electrical conductivity and permittivity.^5,6

Porter et al.⁷ explained that the knowledge of dielectric properties of biological tissues is invaluable and useful in several medical device applications, including cancer detection and treatment. For example, the cell proliferations of breast cancer, oral cancer, cervix cancer, osteosarcoma, and lung carcinoma, as well as intradermal melanoma and intracranial glioma, were successfully inhibited under exposure to intermediate frequency (100, 150, and 200 kHz) and low intensity (200 V/m) alternating electric fields with the duration of exposure to the electric fields for 24 to 72 hours for cell studies, and 10-12 hours per day for 14-21 days and 24 hours for 6 days.^3,8–12 Intermediate frequency electric fields are used to treat cancer because they specifically target cancer cells and do not affect normal cells due to their higher membrane potential than that of cancer cells.^13,14 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.⁸ We used a 100 kHz electric fields because this frequency gave the best results in our in vitro studies, where 28-39% of breast cancer cells died.⁸ Furthermore, we developed non-contact electric fields to avoid dermatitis due to direct contact between the electrodes and the skin, as reported by Kirson et al.³ This novel modality has the potential to reduce 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.¹⁵

Although non-contact electric fields-based therapy has the potential to treat cancer, the safety of such therapy in healthy tissues has to be investigated. This is because injury may occur after exposure to electric fields to organs such as the kidney and liver which have dielectric properties of the kidney and liver, which may interact with electric waves. Therefore, it is important to investigate 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 with a strength of 100 kHz-18 Vpp in the kidney and liver of animal tumour model, with a focus on possible histological alterations in the organs. We hypothesised that exposure to non-contact electric fields would not significantly affect the structure of the kidney and liver. 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 (50-60 V/m) 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 our previously reported study.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 tumour models to study human breast cancer, since it has 98% genetic homology with humans.¹⁶ These rats were provided by the Integrated Research and Testing Laboratory (LPPT) of Universitas Gadjah Mada (UGM), and have never been used for other studies. Rats that were sick or showing symptoms of disorder were excluded from the study. The rats were placed in polypropylene cages for one week of acclimatisation. The polypropylene cage used was communal cage with a size of 50 × 40 cm² and the base was covered with rice hull 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 lamp light, 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 Federer’s 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 the INT and IT groups. The administration of DMBA was conducted twice a week for five weeks. This carcinogenic agent has been widely used in many mammary tumour studies using SD rats.^17,18 Furthermore, the rats in the NIT and IT groups were treated with exposure to intermediate frequency (100 kHz) and low intensity (50-60 V/m) electric fields for 10 hours daily for 21 days in modified individual cages.⁹ Alternating electric fields were generated between pairs of capacitive electrodes embedded in individual cages that have been modified into ECCT devices. ECCT is called non-contact because the electrodes do not stick directly to the animal’s skin. A multidirectional field was generated between pairs of capacitive electrodes and alternated every 0.5 ms (Figure 1). All individual cages were placed on the same table at the same height. The experiment was carried out in a special room which only contained experimental animal cages.⁹

Figure 1. ECCT device for animal study. The size of the cage is 23 cm × 18 cm × 19 cm.

The electrodes are attached to the acrylic wall of the cage with opposite polarity facing each other to produce multiple field directions.

The mammary tumour was palpated every two days with a digital caliper and its size (cm²) was tabulated. Nodule size was not measured in volume due to tool limitation. All tumour measurements were performed by the same investigator (NF). The therapy was terminated once the mammary tumours enlarged to 2.25 cm² in size or therapy was completed on day 21. All rats were returned to their communal cages every day after the therapy was 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 a paint marker as a group marker. Each work in this study, such as DMBA administration, euthanised rat dissection, kidney and liver sample fixation, and data analysis, was carried out by a different investigator. One investigator (FA) controlled and monitored all works in this study.

Results

The result of this study is a comparison of the histological characteristics of the kidney and liver under exposure to non-contact electric fields, which will be coherently described in the sections below.

Histopathology of kidney

The effects of non-contact electric fields exposure on renal histopathology and kidney damages scoring results are illustrated in Figure 2 and Figure 3, respectively. The main damage found in the kidney glomerulus was thickening of Bowman’s capsule, which was significant in all induction groups (p<0.05, 1.16±0.74 for INT, and 1.24±0.59 for IT groups) compared to 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 (p>0.05). In the renal interstitial tissues, inflammation and haemorrhage were identified and the score for both damage in the placebo (NIT) group was the lowest (1.0±0.55) and significantly different from the other three groups (p<0.05, 1.19±0.51 for 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 in the placebo (NIT) group was also the lowest among the treatment groups, but not significantly different from the other three groups (p>0.05).

Figure 2. Histological features of tubular, interstitial, glomerular damages, and congestion in rat kidney sections stained with H&E.

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 3. Scoring of tubular, interstitial, glomerular damages, and congestion in rat kidney sections.

(A) Tubular damage, (B) interstitial damage, (C) glomerular damage, and (D) number of congestions.

Histopathology of liver

The histopathological structure of the liver in the four groups had the same pattern of damage but with different levels of damage as shown in Figure 4 and Figure 5. All groups had the same type of damage, namely cellular damage (pyknosis, karyolysis, karyorrhexis), haemorrhage and congestion, and reversible damage (cellular swelling and fatty change). No significant cellular damage was found in the liver after exposure to non-contact intermediate frequency electric fields (p>0.05). In contrast, the scores of cellular injury and hemorrhage were highest after DMBA administration in INT group (1.96±0.51 and 0.88±0.46, respectively) and significantly different from the control (NINT) group (p<0.05, 1.75±0.43 and 0.63±0.48, respectively). The significant difference in hemorrhage scores between the IT group (p<0.05, 0.87±0.56) and the control (NINT) group (0.63±0.48), due to DMBA administration. 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). The scores of congestion also were not significantly different among groups (p>0.05). The histology of the liver tissue in all groups did not show any 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 scores of congestion among groups and no fibrosis was found, congestion in all groups was still considered normal.

Figure 4. Histological features of haemorrhage, congestion, and cellular damage in rat liver sections stained with H&E.

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 5. Scoring of cellular damage, haemorrhage, and congestion in rat liver sections.

(A) Cellular damage, (B) haemorrhage, and (C) number of congestions.

Discussion

In the present study, the safety of the non-contact intermediate frequency 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 to the glomerulus (Figure 2) may be a result of glomerular hyperfiltration,²⁵ DMBA-induced nephrotoxicity,²⁶ and exposure to electric fields.²⁷ Since significant glomerular damage was observed in 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 Paliwal²⁸ reported that the kidney is one of the main target organs of DMBA (nephrotoxicity) and that epithelial cells of Bowman’s capsule and proximal convoluted tubule appear to be more susceptible to DMBA. Whereas the electric fields affected this damage by changing the transmembrane potential and the distribution of ion channels and dipoles following changes in the membrane structure.²⁹ Although both DMBA administration and non-contact electric field exposure affected the thickening of Bowman’s capsule, the individual toxicity effect of electric field exposure was lower than DMBA administration, as shown in Figure 3. Therefore, DMBA administration would have a greater risk of inducing renal impairment. In our other study using an electric field frequency of 150 kHz with the same intensity (50-60 V/m), thickening of Bowman’s capsule was also found, but DMBA administration and exposure to electric fields did not significantly affect the damage.³⁰

The nephrotoxic effect of DMBA did not only occur in the glomerulus, but also in the tubules. In addition, DMBA caused substantive nephrotoxicity which is characterized by renal tubular necrosis including karyolysis, karyorrexhis and pyknosis,³¹ as shown in Figure 2. Moreover, DMBA created obvious reversible histological changes in the tubules, such as epithelial sloughing and cloudy swelling, as illustrated in Figure 2. Epithelial sloughing represented the progressive tubular disintegration,³² and cloudy swelling may lead to cell necrosis.⁴ However, since the score of each injury to the renal tubules was not significantly different among groups, the nephrotoxic effect of DMBA and the exposure to non-contact electric fields were not harmful to the renal tubules. In our other study using an electric field frequency of 150 kHz with the same intensity (50-60 V/m), DMBA induction significantly damaged renal tubules. This suggests a nephrotoxic effect of DMBA to the renal tubules. In contrast, exposure to this electric field frequency resulted in a decrease in tubular damage in the normal kidneys (NIT group). Moreover, the frequency of this electric field may be able to compensate for tissue repair from the damage caused by DMBA.³⁰ Therefore, exposure to intermediate frequency and low intensity non-contact electric fields was not harmful to the renal tubules. It can even improve the condition of damaged renal tubules.

In the renal interstitial tissue, the nephrotoxic effect of DMBA significantly caused inflammation and haemorrhage, as shown in Figure 3. This inflammation can be affected by oxidative stress and can lead to impaired kidney function, including endothelial dysfunction, atherosclerosis, and glomerular injury.³³ Oxidative stress activates transcription factors including NF-kB, which activates expression of inflammatory response gene.³⁴ In addition, Kandeel et al.³⁵ reported that oxidative stress may alter kidney structure and function due to the effects of reactive oxygen species (ROS) on mesangial and endothelial cells. Oxidative injury happens when ROS, including O₂, H₂O₂ and -OH, ruin the antioxidant defence system of the cells.³⁶ These ROS can be produced due to DMBA administration³⁷ and can spread from their site of production to other sites inside the cell or even prolong the injury outside the cell.³⁸ Moreover, de Oliveira et al.³⁹ revealed that DMBA administration to develop tumours in animal models also causes haemorrhage. In our other study using an electric field frequency of 150 kHz with the same intensity (50-60 V/m), interstitial injury was not significantly induced by DMBA induction or exposure to electric fields.³⁰ 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.⁴⁰ Therefore, exposure to non-contact intermediate frequency electric fields was also not harmful to the renal interstitial tissue. In fact, exposure to this electric field decreased the number of inflammations and haemorrhages in the placebo (NIT) group, as shown in Figure 3.

Almost the same as in the kidney histology, there was no significant damage to the liver after exposure to intermediate frequency non-contact electric fields (Figure 5). The results in the non-therapy (INT) group with the highest scores of hepatocellular damage and haemorrhage indicated that DMBA as a carcinogenic substance can increase the presence of intercellular haemorrhage in the hepatic tissue.²⁴ Duarte et al.²⁴ reported mild hepatotoxicity in the liver, including the presence of a DMBA-induced pyknotic phase of hepatocyte nuclei. However, haemorrhage in the hepatic tissue has not shown symptoms of acute haemorrhage, such as cellular hypoxia, decreased tissue perfusion, organ damage, and death.⁴¹ The results in the therapy (IT) group with a lower hepatocellular damage score compared to the non-therapy (INT) group suggested that exposure to non-contact electric fields had a lower damaging effect than DMBA administration. In addition, since the vascular congestion score was still in a normal condition and not at a chronic level, exposure to non-contact electric fields was not harmful. In our other study using an electric field frequency of 150 kHz with the same intensity (50-60 V/m), hepatocellular damage and congestion were not significantly induced by DMBA induction or exposure to electric fields. Even exposure to this electric field can significantly reduce haemorrhage in the liver of both healthy rats (NIT group) and tumour-induced rats (IT group).³⁰ In another study using exposure to a 150 kHz electromagnetic field, showed mild inflammatory changes with lymphocytic infiltration and haemorrhages in the liver of healthy rats indicating possible liver damage or infection. However, the liver damage that occurred was insufficient to cause clinical and functional manifestations because the lesions were mild enough without significant changes in liver enzyme levels.⁴⁰ Therefore, exposure to intermediate frequency non-contact electric fields was not harmful to the livers of the animals.

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. The thickening of Bowman’s capsule in the NINT group can occur naturally due to aging, or due to ischemia.^30,42 Injury to normal renal tubules can occur because of the high rate of reabsorption by the renal tubules.³⁰ For damage to the renal 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.⁴³ 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.⁴⁴ In addition, the liver itself is a very vulnerable organ due to its size and is the organ most frequently injured after abdominal trauma.⁴⁵

For the results of this study, we only reported the effect of the intermediate frequency non-contact electric field on the histological structure of the kidney and liver, not yet on their function. Renal function parameters such as creatinine and bilirubin, and liver function parameters such as aspartate aminotransferase (AST) and alanine transaminase (ALT) taken from blood serum samples will be reported together with the hematological profile of the rat blood. Based on the evidence of the efficacy and safety of ECCT on normal tissues and organs,^8,9,30 including kidney and liver as reported in this study, we will conduct a phase I clinical trial of ECCT for healthy volunteers using an intermediate frequency (100 kHz) electric field as used in this study. 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.⁴⁶

Conclusions

Exposure to a non-contact electric field with intermediate frequency had a variety of effects on kidney and liver tissues. Together with the effects of DMBA administration, exposure to this electric field can cause thickening of Bowman’s capsule in the glomerulus of the kidney, however, in other kidney tissues and in the liver, exposure to this electric field was safe. In addition, individual exposure to this electric fields did not cause significant thickening of the Bowman’s capsule. It can even decrease the number of inflammations and haemorrhages in the organs.

Data availability