Radioprotective Effects of Honeybee Venom ( Apis mellifera ) Against 915-MHz Microwave Radiation–Induced DNA Damage in Wistar Rat Lymphocytes: In Vitro Study

Abstract

The aim of this study is to investigate the radioprotective effect of bee venom against DNA damage induced by 915-MHz microwave radiation (specific absorption rate of 0.6 W/kg) in Wistar rats. Whole blood lymphocytes of Wistar rats are treated with 1 μg/mL bee venom 4 hours prior to and immediately before irradiation. Standard and formamidopyrimidine-DNA glycosylase (Fpg)–modified comet assays are used to assess basal and oxidative DNA damage produced by reactive oxygen species. Bee venom shows a decrease in DNA damage compared with irradiated samples. Parameters of Fpg-modified comet assay are statistically different from controls, making this assay more sensitive and suggesting that oxidative stress is a possible mechanism of DNA damage induction. Bee venom is demonstrated to have a radioprotective effect against basal and oxidative DNA damage. Furthermore, bee venom is not genotoxic and does not produce oxidative damage in the low concentrations used in this study.

Apis mellifera venom is a complex mixture of at least 18 active components including peptides, enzymes, and amines, which have a wide variety of pharmaceutical properties. The major components of bee venom are melittin, phospholipase A₂, apamin, adolapin, histamine, catecholamines, and mast cell degranulating peptide. ^1–3 Melittin, the main component and principal toxin in bee venom, comprising 40% to 50% of the dry weight of the venom, ⁴ is a highly basic polypeptide consisting of 26 amino acid residues. ^1,2,5,6 Melittin is mostly hydrophobic but has a hydrophilic sequence and is known to damage cell membrane enzyme system. ¹ It is also known to have effects on erythrocytes, leukocytes, thrombocytes, and many pharmacological systems. ^1,7

Bee venom is used in traditional medicine to treat a variety of conditions, such as arthritis, rheumatism, back pain, and skin disease. ^8,9 In recent years it has been reported that bee venom possesses antimutagenic, ¹⁰ proinflammatory, ¹¹ anti-inflammatory, ¹² antinociceptive, ¹³ and anticancer effects. ¹⁴ Recent studies also reported additional effects of bee venom such as induction of apoptosis and necrosis, effects on proliferation and growth inhibition, and cytotoxic effects on different types of cancer cells. ^15–21

In addition to the wide range of the bee venom’s activities, it possesses a radioprotective capacity that was noted against X ray and gamma radiation in various test systems. ^22–26 There is increased use of nonionizing radiation in industry, commerce, medicine, and the home, especially in mobile telephones. Although the average exposure levels are low compared with exposure limits, there is an enhanced possibility of adverse effects of non-ionizing radiation to humans. Because of that, there has been a growing concern among the public regarding the potential human health hazard of exposure to these frequencies by these devices. ^27–30

Cytogenetic studies of microwave radiation were conducted in vitro as well as in vivo and yielded contradictory and often intriguing results. ^31–34 Some of the published reports suggested that exposure of human cells and animals to radiofrequency radiation does not result in increased cytogenetic damage. ^35–42 On the other hand, a range of studies have shown positive results stating that radiofrequency radiation can indeed induce genetic alternation after exposure to electric field. ^43–52

A combination of different methods may play an important role in the assessment of possible genotoxic damage caused by microwave radiation. The comet assay is now a well-established genotoxicity test for the estimation of DNA damage at the individual cell level both in vivo and in vitro. The comet assay has been widely used to quantify DNA damage in lymphocytes from subjects exposed to several environmental or occupational substances and especially to estimate oxidative damage to DNA. ^53–56 In combination with certain bacterial enzymes that recognize oxidized purines and pyrimidine bases, this assay has been used to determine oxidative DNA damage that has been implicated in several health conditions. ^54,57–61 Therefore, this method makes it possible to evaluate the level of primary DNA damage even after short-term exposure to radiation.

Because of the lack of conclusive data on the genotoxic status induced by microwave radiation, the aim of this study was assessment of the radioprotective effect of whole bee venom against 915-MHz microwave radiation–induced DNA damage in the Wistar rat’s peripheral blood lymphocytes in vitro. The possible genotoxic effect of bee venom alone was also assessed on nonirradiated lymphocytes. For that purpose, the alkaline comet assay was used as a sensitive tool for detection of DNA damage. In addition to the standard protocol for the comet assay, the formamidopyrimidine- DNA glycosylase (Fpg)–modified comet assay was used as a much more sensitive and specific technique for detection of DNA strand breaks and oxidative stress.

Materials and Methods

Animals

Animal studies were carried out according to the guidelines of the Republic of Croatia ⁶² and in compliance with the US Guide for the Care and Use of Laboratory Animals. ⁶³ Adult male Wistar rats (11 weeks old, approximate body weight 350 g) were used in this study. The animals had passed through an accommodation period of 1 week. The animals were kept in steady-state microenvironment conditions (22 ± 1°C) and received standard laboratory food and water ad libitum, with alternating 12-hour light and dark cycles.

Blood Sampling

The whole blood samples (8 mL) were collected by cardiac puncture under sterile conditions in heparinized vacutainer tubes (Becton Dickinson, Franklin Lakes, New Jersey) containing lithium heparin as anticoagulant. After collection, blood was divided into 1-mL aliquots and placed into 24-well culture plates according to the exposure conditions. All experiments were conducted on peripheral blood lymphocytes cultivated at 37°C in an atmosphere of 5% CO₂ in air.

Bee Venom (Apis mellifera)

Lyophilized whole bee venom was purchased from Sigma (St Louis, Missouri). Just before the beginning of the experiment, bee venom was dissolved in 1 mL sterile redistilled water at 25°C and centrifuged at 12 000 rpm for 10 minutes to remove insoluble materials. Bee venom was added to lymphocyte cultures in final concentration of 1 μg/mL, 4 hours prior to irradiation and immediately before irradiation. To test whether this concentration alone induces genotoxic effect, lymphocytes were treated under the same conditions with bee venom in corresponding time periods. The concentration of 1 μg/mL was noncytotoxic toward Wistar rat lymphocytes after evaluation of cytotoxicity of the large number of concentrations ranging from 0.1 to 10 μg/mL (unpublished data).

Exposure Conditions

An electromagnetic field was generated within the certified gigahertz transversal electromagnetic mode (GTWM) cell manufactured by ETS Lindgren (model 5402, ETS Lindgren, St Louis, Missouri). A signal generator was used to produce electromagnetic field frequency of 915 MHz (Anritsu 27211A, Tokyo, Japan). A signal amplifier (RF 3146 Power Amp Module RF Micro Devices, Greensboro, North Carolina) and signal modulator (RF 2722 Polaris chip, RF Micro Devices) were part of the exposure setup. The signal amplifier was used to amplify RF signal induced by signal generator, whereas the signal modulator was used to modulate continuous wave to a signal used in global system mobile (GSM) phones. Whole blood was exposed to the carrier frequency of 915 MHz with GSM basic signal modulation for 30 minutes. Incident electromagnetic field strength of 30 V/m was uniform over the entire biological object throughout the exposure procedure. The power density of the field was 2.4 W/m², corresponding to approximate wholebody specific absorption rate (SAR) of 0.6 W/kg. ⁶⁴

Alkaline Comet Assay

The comet assay was carried out under alkaline conditions, as described by Singh et al. ⁵³ Fully frosted slides were covered with 1% normal melting point (NMP) agarose (Sigma). After solidification, the gel was scraped off the slide. The slides were then coated with 0.6% NMP agarose. When this layer had solidified, a second layer containing 5 μL of whole blood sample mixed with 0.5% low melting point (LMP) agarose (Sigma) was placed on the slides. After 10 minutes of solidification on ice, the slides were covered with 0.5% LMP agarose. The slides were then immersed for 1 hour in ice-cold freshly prepared lysis solution (2.5 M NaCl, 100 mM disodium EDTA, 10 mM Tris-HCl, 1% sodium sarcosinate [Sigma], pH 10, with 1% Triton X-100 [Sigma] and 10% dimethyl sulfoxide [Kemika, Zagreb, Croatia]; added immediately prior to use) to lyse the cells and allow DNA unfolding. The slides were then placed on a horizontal gel electrophoresis tank, facing the anode. The unit was filled with fresh electrophoresis buffer (300 mM NaOH, 1 mM disodium EDTA, pH 13.0) and the slides were placed in this alkaline buffer for 20 minutes to allow DNA unwinding and expression of alkali-labile sites. Denaturation and electrophoresis were performed at 4°C under dim light. Electrophoresis was carried out for 20 minutes at 25 V (300 mA). After electrophoresis, the slides were rinsed gently 3 times with neutralization buffer (0.4 M Tris-HCl, pH 7.5) to remove excess alkali and detergents. Each slide was stained with ethidium bromide (20 μg/mL) and covered with a cover slip. The slides were then stored in sealed boxes at 4°C until analysis.

Fpg-modified Comet Assay

The analysis of 8-oxo-7, 8-dihydro-2′-deoxyguanozine (8-oxodG) was performed using an Fpg FLARE assay kit (Trevigen Inc, Gaithersburg, Maryland) with some modification. Fully frosted microscopic slides were prepared. Each slide was covered with 1% NMP agarose (Sigma). After solidification, the gel was scraped off the slide. The slides were then coated with 0.6% NMP agarose. An LMP agarose was melted and stabilized in a water bath at 37°C. For each sample and control, 5 μL of whole blood was mixed with 100 μL of LMP agarose (provided with the FLARE^™assay kit) and placed on the slides. After 10 minutes of solidification on ice, the slides were covered with 0.5% LMP agarose. The slides were then immersed in a prechilled lysis solution (provided with the FLARE^™ assay kit) and kept in a refrigerator at 2°C for 60 minutes followed by immersion in the FLARE buffer, 3 times for 15 minutes. After lysis, the slides were treated with 100 μL of Fpg enzyme (1:500 in REC dilution buffer, provided with the FLARE assay kit). The enzyme was diluted right before use. Control slides were treated with 100 μL of REC dilution buffer only. The slides were placed horizontally in a humid chamber at 37°C for 30 minutes. All slides were then immersed in an alkali solution (0.3 M NaOH, 1 mM Na₂ EDTA, pH 12.1) for 40 minutes followed by electrophoresis in a prechilled alkali solution (0.3 M NaOH, 1 mM Na₂ EDTA, pH 12.1) at 1 V/cm for 20 minutes. After electrophoresis, the slides were rinsed gently 3 times with neutralization buffer (0.4 M Tris-HCl, pH 7.5) to remove excess alkali and detergents. Each slide was stained with ethidium bromide (20 μg/mL) and covered with a cover slip. Slides were stored at 4°C in sealed boxes until analysis.

Comet Capture and Analysis

A total of 100 randomly captured cells from each slide was examined at 250× magnification using an epifluorescence microscope (Zeiss, Oberkochen, Germany) connected through a black and white camera to an image analysis system (Comet Assay II; Perceptive Instruments Ltd, Haverhill, Suffolk, UK). A computerized image analysis system was used to acquire images, compute the integrated intensity profile for each cell, estimate the comet cell component, and evaluate the range of derived parameters. To quantify DNA damage, the following comet parameters were evaluated: tail length (μm), tail intensity (%DNA), and tail moment. Tail length (ie, the length of DNA migration) is related directly to the DNA fragment size and is presented in micrometers. It was calculated from the center of the cell. Tail moment was calculated as (tail length × percentage of DNA in tail)/100. The analysis did not include the edges and damaged parts of the gel or debris, superimposed comets, and comets without distinct head (“clouds,” “hedgehogs,” or “ghost cells”).

Differences in the tail length, tail intensity, and tail moment between samples obtained with standard alkaline comet assay (basic DNA damage) and Fpg-modified comet assay (total DNA damage) were considered as 8-oxodG (oxidative DNA damage) in a single cell.

Statistical Analysis

Each experimental set contained duplicate slides. The various parameters (tail length, tail intensity, and tail moment) measured in the exposed and control groups were evaluated using Statistica 5.0 package (StaSoft, Tulsa, Oklahoma). Each sample was characterized for the extent of DNA damage by considering the mean ± standard error of the mean (SE), median and range of the comet parameters. To normalize distribution and to equalize the variances, a logarithmic transformation of data was applied. Multiple comparisons between groups were done by means of analysis of variance (ANOVA) on log-transformed data. Post hoc analysis of differences was done by Scheffé test. The level of statistical significance was set at P < .05.

Results

Table 1 shows the results of the alkaline comet assay. Mean values ± SE of the standard comet tail lengths were 12.51 ± 0.07 μm for the control samples and 13.38 ± 0.11 μm for the samples exposed to microwave radiation (MW) for 30 minutes. Mean values ± SE for bee venom (BV)–treated samples were from 12.52 ± 0.08 μm to 12.56 ± 0.08 μm depending on the time of treatment and from the 12.85 ± 0.07 μm to 12.91 ± 0.10 μm for the irradiated samples and corresponding BV treatment. Mean tail intensity was 0.88 ± 0.18 for the control samples and 0.59 ± 0.09 for the samples exposed to MW for 30 minutes. Mean values ± SE for BV-treated samples ranged from 0.59 ± 0.08 to 0.75 ± 0.14 depending on the time of treatment and from 0.62 ± 0.09 to 0.66 ± 0.15 for the irradiated samples and corresponding BV treatment. Mean comet moment was 0.10 ± 0.02 μm for the control samples and 0.07 ± 0.01 μm for the samples exposed to MW for 30 minutes. Mean values ± SE for BV-treated samples were from 0.07 ± 0.01 μm to 0.09 ± 0.02 μm depending on the time of treatment and 0.07 ± 0.01 μm for the irradiated samples and corresponding BV treatment.

Table 2 shows the results of the Fpg-modified comet assay. As for the Fpg-modified comet parameters, tail lengths were 14.97 ± 0.17 μm for the control samples and 17.51 ± 0.22 μm for the samples exposed to MW for 30 minutes. Mean values ± SE for BV-treated samples were from 14.76 ± 0.15 μm to 14.82 ± 0.21 μm depending on the time of treatment and from 14.33 ± 0.15 μm to 14.51 ± 0.16 μm for the irradiated samples and corresponding BV treatment. Mean tail intensity was 2.92 ± 0.36 for the control samples and 4.82 ± 0.53 for the samples exposed to MW for 30 minutes. Mean values ± SE for BV-treated samples were from 2.36 ± 0.27 to 2.73 ± 0.33 depending on the time of treatment and from 1.97 ± 0.32 to 2.58 ± 0.22 for the irradiated samples and corresponding BV treatment. Mean comet moment was 0.37 ± 0.04 μm for the control samples and 0.66 ± 0.07 μm for the samples exposed to MW for 30 minutes. Mean values ± SE for BV-treated samples were from 0.30 ± 0.03 μm to 0.35 ± 0.04 μm depending on the time of treatment and from 0.23 ± 0.04 μm to 0.31 ± 0.03 μm for the irradiated samples and corresponding BV treatment.

Basic statistics for the parameters of standard and Fpg-modified comet assay for different time of exposure are presented in Tables 1 and 2. According to 1-way ANOVA, only the parameter for tail length showed significant deviation from normal distribution in the alkaline comet assay. In the Fpg-modified comet assay, all 3 parameters (tail length, tail intensity, and tail moment) showed significant deviation from normal distribution for the irradiated sample. When we compared mean values between different samples in both comet tests, the difference was higher when the Fpg-modified comet assay was applied.

According to the 1-way ANOVA, only the 3 parameters of the Fpg-modified comet assay showed statistically significant differences between the irradiated group compared with the control group, the BV treatment group, and the MW/BV treatment group.

When the standard comet assay was applied, the mean tail length for irradiated sample differed significantly from nontreated control samples, BV-treated samples, and MW/BV-treated samples. For tail intensity and tail moment, no significant difference was found between the control sample and the samples treated with only BV or with MW/BV. In the Fpg-modified comet assay, all 3 parameters differed significantly from nontreated control samples, BVtreated samples, and MW/BV-treated samples. Generally, the means and medians of all 3 standard comet assay parameters were lower than those of the Fpg-modified comet assay. These findings suggest that the Fpg-modified comet assay is more sensitive to the effects of microwave radiation than the standard comet assay.

Discussion

In the past few decades, many publications regarding health hazards of microwave radiation have appeared in the scientific literature. Many of these studies disagree and yield contradictory results about the effects of microwave radiation on human health and especially on the genetic material of the cell. Given the growing use of cellular phones, the proportion of the population exposed to this type of radiation is increasing. Added to this group are occupationally exposed personnel, demonstrating the importance of studying the possible health risk of this type of radiation. If microwave radiation indeed carries risks, there is a need to find a radioprotective agent that is nontoxic to the cells. Many of the chemicals that have been demonstrated to have radioprotective effects also have unacceptable toxic side effects. ^65,66 Investigations to find noncytotoxic and effective compounds have led to an increasing interest in natural products, including animal venoms. ^23,67,68

This investigation tested whether bee venom possesses radioprotective effects against microwave radiation. In previous studies, the venom of Apis mellifera was determined to be radioprotective for X ray and gamma radiation. ^22,24–26

In the study by Kanno et al, ²³ mice were subcutaneously injected with 2.8 or 5.6 μg/g of body weight of bee venom prior to whole-body exposure to 937 R of gamma radiation from cobalt-60. The nontreated irradiated mice demonstrated 100%mortality 15 days after irradiation. Survival to the 60th day post irradiation was 10% for the group that received 2.8 μg bee venom per gram of body weight and of 18% for the group that received 5.6 μg bee venom per gram of body weight. In the animals treated only with the bee venom, direct toxic effects caused by the venom were not observed.

According to the Shipman and Cole, ²² bee venom belongs to a class of radioprotectors known as biostimulants, because it alters the body’s physiological state. For this reason, bee venom is effective only when injected 1 day before irradiation. These authors tested the radioprotective property of bee venom administered subcutaneously in concentrations from 1.1 to 5.6 μg/g in mice 24 hours prior to irradiation with a lethal dose of X rays (800 R, 825 R, and 850 R). The authors observed a significantly higher number of surviving animals, with a survival rate of 64%, 70%, and 80% in 30 days. The same authors also reported that bee venom might produce stress in animals and thereby elicit the so-called adaptation syndrome, which would increase radioresistance.

Varanda et al ²⁴ demonstrated the radioprotective property of whole bee venom when administered intraperitoneally 24 hours prior to exposure to a dose of 3.0 Gy of gamma rays, analyzing the number of chromosomal aberrations detected in bone marrow cells of Wistar rats. These authors noticed a significant decrease in the total number of aberrations, from 46.8% in the group that only received radiation to 14.4% in the group that was exposed to radiation and received bee venom (0.5 μL/100 g weight and 1 μL/100 g weight), presenting a protection magnitude around 70%. Additionally, the authors observed a reduction in the number of cells with chromosomal aberrations from 22.4% in the group exposed only to radiation to 9.5% in the group that received bee venom (0.5 μL/100 g weight and 1 μL/100 g weight) 24 hours prior to radiation.

The radioprotective effect of bee venom was also noted by Varanda and Takahashi ²⁵ in in vitro studies with human peripheral blood lymphocytes. Bee venom protected against the induction of dicentric chromosomes with 2.0 Gy gamma radiation when cultures were treated with 0.00015 μL venom/l mL medium 6 hours before irradiation. The authors stated that this protection may be attributed to a stimulation of the cell repair system. Within this context, the venom of Apis mellifera is believed to have radioprotective activity attributable mainly to the stimulation of the hematopoietic system. However, other mechanisms such as release of histamine induced by the mast cell degranulating (MCD) peptide and reduction in blood oxygen tension induced by phospholipase A2 contribute to the radioprotective effect of bee venom. ²⁶ It was also clear that the venom itself has no clastogenic activity, as demonstrated by the fact that it did not induce an increase in the frequency of sister chromatid exchanges (SCE) at the concentration used. ²⁵

In our study, the alkaline comet assay and its Fpgmodification were used as 2 sensitive methods for detecting DNA damage after exposure to microwave radiation. Based on the results of this study using the standard version of comet assay, microwave radiation increased DNA damage in Wistar rat lymphocytes. Treatment with bee venom prior to radiation exposure and immediately before irradiation protected against significant DNA damage. Bee venom administered alone at a concentration of 1 μg/mL also did not have any impact on genotoxicity of Wistar rat lymphocytes in vitro.

The difference in measured DNA damage between the standard and the Fpg-modified comet assays indicates the presence of 8-oxodG as the result of oxidative DNA damage. Specific information about oxidative DNA damage and repair can be obtained by modifying the standard comet assay including Fpg, a restriction enzyme that recognizes and removes oxidized purines and some alkylated DNA products. This endonuclease is involved in the first step of the base excision repair to remove specific modified bases from DNA and creates apurinic or apyrimidinic (AP) sites that are subsequently cleaved by AP lyase activity, creating a gap in the DNA strand. These lesions are measured as additional strand breaks that can be detected by the comet assay. ^54–57 To determine whether microwave radiation–induced DNA damage is mediated by oxidative stress, the standard comet assay was combined with the Fpg-modified version, indicating significant levels of Fpg-sensitive sites, which is indicative of oxidative DNA-base modification. In the present study, the difference between the comet assay results in the presence and in the absence of Fpg-enzyme suggests that oxidative stress is responsible for the DNA damage induced by microwave radiation. However, basal DNA damage detected by the standard version of the comet assay indicates not only that DNA damage is the result of oxidative stress but also that some other mechanism of genotoxicity is involved as well.

The Fpg-modified comet assay also served as a sensitive tool for assessment of radioprotective effect of bee venom against genotoxic effect of microwave radiation. When bee venom was administered 4 hours prior to and immediately before irradiation, all 3 parameters of Fpg-modified comet assay—tail length, tail intensity, and tail moment—showed a decrease in DNA damage compared with the irradiated sample only but did not show any significant increase compared with control. Apis mellifera venom alone did not show any genotoxic effect toward Wistar rat lymphocytes in the concentrations used with Fpg-modified protocol.

Reports in the literature have assessed potential health risks of microwave radiation attributable to oxidative stress. Stopczyk et al ^69,70 showed that the 900-MHz electromagnetic field produced by mobile phones affects the activity of superoxide dismutase and the level of malondialdehyde in human blood platelets. The authors concluded that oxidative stress after exposure to microwaves may be the reason for many adverse changes in human blood platelets.

Köylü et al ⁷¹ stated that microwaves (MW) from cellular phones may affect biological systems by increasing free radicals, which may enhance lipid peroxidation levels of the brain, thus leading to oxidative damage. Their study was designed to determine the effects of MW radiation on the brain lipid peroxidation system and the possible protective effects of melatonin on brain degeneration induced by MW. Male rats were randomly divided into 3 groups: a group exposed to 900 MHz alone, a group exposed to 900 MHz and melatonin, and an unexposed control group. After treatment, brain cortex and hippocampus were removed to study the levels of lipid peroxidation as malondialdehyde. The levels of lipid peroxidation in both brain locations increased in the MW radiation group compared with the control group, although the levels in the hippocampus were decreased by MW radiation and melatonin administration. The brain cortex lipid peroxidation levels were unaffected by melatonin treatment. The authors concluded that melatonin may prevent MW-induced oxidative changes in the hippocampus by strengthening the antioxidant defense system, thereby reducing oxidative stress products.

Yurekli et al ⁷² investigated 945-MHz frequency radiation and effects on oxidative stress in rats. In this study, a GTEM cell was used as an exposure environment for plane wave conditions of far-field free space electromagnetic field propagation at the GSM base transceiver station frequency of 945 MHz. When electromagnetic fields were applied at a power density of 3.67 W/m² (SAR of 11.3 mW/kg), which is well below current exposure limits, malondialdehyde level was found to increase and glutathione concentration was found to decrease significantly. Additionally, there was a less significant increase in superoxide dismutase activity demonstrating induction of oxidative stress under electromagnetic exposure.

All of these findings, in addition to our results gained by the Fpg-modified comet assay, suggest that oxidative stress can cause DNA damage in Wistar rat lymphocytes in addition to some basal DNA damage detected with the standard comet assay after exposure to 915-MHz microwave radiation in vitro. Bee venom from Apis mellifera in this study demonstrated radioprotective effects against low-level microwave radiation when administered prior to the radiation exposure and immediately before irradiation. This study also demonstrated that bee venom has a radioprotective effect against basal and oxidative DNA damage. Furthermore, bee venom itself has no effect on induction of DNA breaks or on oxidative stress in the low concentrations used in this research. A combination of the standard and the Fpg-modified comet assay has proven useful in detecting genotoxicity of microwave radiation and radioprotection of bee venom.

The ever-increasing use of cellular phones and the number of associated base stations are becoming a widespread source of nonionizing electromagnetic radiation. Within this context, the venom of Apis mellifera could be considered as an effective and nontoxic radioprotector.