|Year : 2018 | Volume
| Issue : 2 | Page : 89-98
Inhibitory effect of bee venom against potassium bromate causing genetic toxicity and biochemical alterations in mice
Abeer H Abd El-Rahim, Omaima M Abd-El-Moneim, Heba A.M. Abd El-Kader, Amira Abd El Raouf
Department of Cell Biology, National Research Centre, Dokki, Giza, Egypt
|Date of Submission||03-Aug-2018|
|Date of Acceptance||02-Sep-2018|
|Date of Web Publication||28-Dec-2018|
Dr. Abeer H Abd El-Rahim
Department of Cell Biology, National Research Centre, Dokki, Giza 12922
Source of Support: None, Conflict of Interest: None
Background/aim Bee venom (BV) therapy is a highly effective treatment, capable of improving one’s health. The present study attempts to assess the effect of BV on the toxicity of oral administration of potassium bromate (KBrO3) which has been widely used in food and cosmetic industries.
Materials and methods Sixty adult male mice were gavaged with KBrO3 at two doses (100 and 200 mg/kg body weight) for 10 days. Afterwards, BV at a dose of 120 μg/kg body weight was injected subcutaneously three times per week for two successive weeks. The genetic study was performed using chromosomal aberration and micronucleus formation in the bone marrow, DNA fragmentation in liver cells and by sperm analysis. In addition, serum biochemical markers such as catalase and malondialdehyde, kidney, and liver functions were assessed.
Results The results have shown that KBrO3 caused DNA damage that represented the increase in the frequencies of chromosome abnormalities, micronuclei formation, percentage of DNA fragmentation, and sperm morphological abnormalities. Meanwhile, the results showed that KBrO3 exhibited severe toxicity for antioxidant activities for liver and kidney functions. Conversely, BV significantly decreased the frequencies of DNA damage in all aforementioned parameters induced by KBrO3. In addition, it improved the antioxidant activities and the function of the liver and kidneys.
Conclusion BV has a potent ameliorating effect against the KBrO3 hazard impacts in animal tissues especially at higher doses. This observation indicated that BV could be a potential therapeutic agent in the treatment of KBrO3 risk.
Keywords: bee venom, chromosome aberrations, DNA fragmentation, liver and kidney functions, mice, micronucleus test, potassium bromate
|How to cite this article:|
Abd El-Rahim AH, Abd-El-Moneim OM, Abd El-Kader HA, Abd El Raouf A. Inhibitory effect of bee venom against potassium bromate causing genetic toxicity and biochemical alterations in mice. J Arab Soc Med Res 2018;13:89-98
|How to cite this URL:|
Abd El-Rahim AH, Abd-El-Moneim OM, Abd El-Kader HA, Abd El Raouf A. Inhibitory effect of bee venom against potassium bromate causing genetic toxicity and biochemical alterations in mice. J Arab Soc Med Res [serial online] 2018 [cited 2019 Apr 25];13:89-98. Available from: http://www.new.asmr.eg.net/text.asp?2018/13/2/89/248985
| Introduction|| |
Potassium bromate (KBrO3) is used in making bread for giving strength and elasticity to the dough during the baking process. It is also used as food additive and in cheese production, beer making, and is commonly added to fish paste products . It is also important in pharmaceutical and cosmetic industries and is a constituent of cold wave hair solutions . In addition, KBrO3 found as a byproduct of ozone disinfection in drinking water samples .
KBrO3 has been classified by the International Agency for Research on Cancer as a carcinogenic compound ,, where it causes renal tumors , mesotheliomas of the peritoneum, and follicular cell tumors of the thyroid in rats . Administration of KBrO3 to rats was found to induce oxidative stress and reduced the antioxidant power of rat blood . KBrO3 is highly injurious to tissues especially the central nervous system tissues . It causes primary DNA oxidative damage and increases 8-hydroxydeoxyguanosine DNA adduct levels, a representative marker of oxidative DNA modification, in vivo and in vitro . It also caused structural chromosomal aberrations in the bone marrow cells of rats . In addition, KBrO3 caused micronuclei in peripheral blood reticulocytes in mice and rats , and in the cells of the glandular part of stomach and liver of rats .
Bee venom (BV) is a natural toxin produced by the honey bee and it has a prime role of defense for the bee colony and protects bees from external predators by a complex mixture of substances ,. It contains different peptides including melittin, apamin, adolapamin, and mast cell degranulating peptide ,; the major component is melittin which is about 50% of its dry weight . BV also contains biologically active amines, nonpeptide components as well as enzymes, which is composed of hyaluronidase acid, phosphomonoesterase, α-D-glucosidase, phospholipase A2, and lysophospholipase . Many recent studies have demonstrated that BV has radioprotective , antimutagenic , anti-inflammatory , antinociceptive , and anticancer , activities on various types of cancerous cells, such as the liver , bone , prostate, breast, cervical, and renal cancer cells ,. Therefore, in recent years the popularity of the BV as an alternative therapy in the treatment of many diseases has been increased.
The present work aims to assess the effect of BV on genetic toxicity and biochemical alterations induced by KBrO3 in mice. The genetic study will include chromosomal aberration and micronucleus assays in the bone marrow, DNA fragmentation in liver cells, and sperm physical characters. In addition, the biochemical study will include assessment of liver and kidney functions as well as the malondialdehyde (MDA) content and catalase (CAT) enzyme in albino mice.
| Materials and methods|| |
BV obtained from VACSERA Co (Dokki, Cairo, Egypt).
KBrO3 obtained from El-Gomhouria Company (Cairo, Egypt), in the form of a white powder.
Male Swiss albino mice (Mus musculus) 3 months of age, weighing 25–30 g were obtained from the National Research Centre (Cairo, Egypt). These mice were maintained under controlled conditions of temperature (25±2°C) and light (12 light : 12 dark). Mice were provided with standard diet and tap water. They were acclimatized to laboratory conditions for at least 7 days before the experiment.
All animals received human care in compliance with the guidelines of Animal Care and Use Committee of the National Research Centre and the National Institute of Health (NIH publication 86-23 revised 1985).
This experiment was conducted on 60 Swiss albino mice divided equally into six groups.
- Control group (group 1): this group served as a negative control group and received saline solution at a dose of 120 μg/kg body weight.
- BV group (group 2): these animals were subcutaneously injected with BV at a dose of (120 μg/kg body weight) three times per week for two successive weeks according to Alvarez-Fischer et al .
- Low-dose KBrO3 group (group 3): these animals were injected with KBrO3 at a dose of 100 mg/kg body weight orally for 10 days.
- High-dose KBrO3 group (group 4): these animals were orally gavaged KBrO3 (200 mg/kg body weight) for 10 days. The doses of KBrO3 used in the current study were adjusted according to Kurokawa et al. .
- Low-dose KBrO3+BV group (group 5): animals orally gavaged KBrO3 as in group 3, then BV was injected subcutaneously at a dose of 120 μg/kg body weight three times per week for two successive weeks.
- High-dose KBrO3+BV group (group 6): animals received a high dose of KBrO3 orally as in group 4, and then the BV was injected subcutaneously at a dose of 120 μg/kg body weight three times per week for two successive weeks.
At the end of each experiment two portions of blood samples were collected, one in heparinized tubes to obtain plasma and the other portion was allowed in tubes for 30 min to clot and centrifuged at 5000 rpm for 10 min to obtain serum for biochemical analysis. The bone marrow and liver samples were collected for genetic analysis; moreover, sperms of mice were collected from the epididymis, then prepared and analyzed for sperm count and morphology.
Chromosomal aberration assay
Chromosomal aberration assay was performed as per the method of Schmid  and modified by Aron et al. . Mice were sacrificed by cervical dislocation and the bone marrow was obtained from both femora. Routine preparations of metaphases were made and stained with Giemsa. Chromosomal aberration was calculated by counting 50 metaphase spreads/animal.
The micronucleus assay was carried out in bone marrow, according to the method of Schmid . A total of 1000 polychromatic erythrocytes were scored per animal for the evaluation of the frequencies of micronucleated polychromatic erythrocytes.
Sperms were prepared and analyzed according to the protocols of Wyrobek and Bruce .
A drop of sperm suspension was smeared onto a slide, left to dry and then stained with eosin A; the slides were washed in water and air dried again. The smears were microscopically analyzed at a magnification of ×1000 for observation of abnormalities .
Sperm counts were performed visually using a hamocytometer. The count was repeated three times for each sample to minimize error .
DNA fragmentation assay
Apoptotic DNA fragmentation was qualitatively analyzed by detecting the laddering pattern of nuclear DNA according to Lu et al. . In brief, liver tissues were homogenized, washed overnight at 37°C in PBS, and lysed in 0.5 ml of DNA extraction buffer (50 mM Tris-HCl, 10 mM EDTA. 0.5% Triton, and 100 µg/ml proteinase K, pH 8.0). The lysate was then incubated with 100 µg/ml DNase-free RNase for 2 h at 37°C, followed by three extractions of an equal volume of phenol/chloroform (1 : 1 v/v) and a subsequent re-extraction with chloroform by centrifuging at 15 000 rpm for 5 min at 4°C. The extracted DNA was precipitated in two volume of ice-cold 100% ethanol with 1/10 volume of 3 M sodium acetate, pH 5.2 at −20°C for 1 h, followed by centrifuging at 15 000 rpm for 15 min at 4°C. After washing with 70% ethanol, the DNA pellet was air dried and dissolved in 10 mM Tris-HCl/1 mM EDTA, pH 8.0. The DNA was then electrophoresed on 1.5% agarose gel and stained with ethidium bromide in Tris/acetate/EDTA (TAE) buffer (pH 8.5, 2 mM EDTA, and 40 mM Tris-acetate). A 100-bp DNA ladder (Invitrogen, Waltham, Massachusetts, USA) was included as a molecular size marker and DNA fragments were visualized and photographed by exposing the gels to ultraviolet transillumination.
The quantitative measurements of MDA and CAT enzyme were done in the plasma according to the method of Ohkama et al.  and Aebi , respectively, using kits of Biodiagnostic Co. (Dokki, Giza, Egypt).
The kidney function was measured in the serum to determine the levels of urea according to Bartles and Bohmer  and creatinine according to Fowcelt and Scott , using kits of Biodiagnostic Co.. The estimation of the levels of glutamic oxaloacetic transaminase and glutamic pyruvate transaminase activities were done according to IFCC , the protein determined according to Gornall et al. . In addition, the determination of total bilirubin was performed according to Water and Gerade  and alkaline phosphatase according to the Scandinavian Society  using kits of Biodiagnostic Co..
Statistical analysis was carried out with statistical package for the social sciences software, version 22 (IBM SPSS). Data were analyzed using one-way analysis of variance followed by Duncan’s multiple test at a significant level of P value less than or equal to 0.05. The data are represented by mean±SE.
| Results|| |
The results regarding the protective effects of BV against the KBrO3-induced chromosomal aberrations in male mice are summarized in [Table 1]. There were many different structural chromosomal aberrations such as gap, break, deletion, centric fusion, centromeric attenuation, and endomitosis in addition to numerical aberrations such as aneuploidy and polyploidy. Our data has shown that there was no significant difference in the frequencies of chromosomal aberrations between BV and the negative control group for most type of aberrations. Low and high doses of KBrO3 showed a significant increase (P≤0.05) in chromosomal aberrations comparing with vehicle control. In contrast, a significant reduction in chromosomal aberrations was observed in animals treated with BV after KBrO3 and tends to control in most aberrations in low dose. Aberrations in high dose also decreased but did not reach the control group.
|Table 1 Effect of potassium bromate and bee venom on chromosomal aberrations of mice bone marrow|
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[Figure 1] represents the mouse bone marrow micronucleus test (×1000) with Giemsa stain showing none and micronucleated polychromatic erythrocytes. The result has shown that there was no statistically significant difference in the number of micronuclei between BV and control groups. The number of micronuclei significantly increased (P≤0.05) by treatment with low and high doses of KBrO3 compared with the control group. In addition, there was a marked increase in the number of micronuclei in high dose of KBrO3 than low dose. In contrast, groups treated with KBrO3 followed by BV injection showed a significant decrease (P≤0.05) in the micronucleus frequency compared with KBrO3-treated groups ([Figure 2]).
|Figure 1 Photograph of mouse bone marrow micronucleus test. Arrow (a) represents normal cell and arrow (b) represents micronucleated polychromatic erythrocytes.|
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|Figure 2 Effect of potassium bromate and bee venom on the frequencies of micronucleated polychromatic erythrocytes (MNPCE) in male mice. Values with different letters are significantly different at P value less than or equal to 0.05.|
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Sperm morphology and count
The results of sperm morphology analysis are presented in [Table 2]. There are different types of sperm abnormalities included amorphous, without hock, small, big, and banana heads, in addition to coiled tail. There was no significant difference between BV and the control group in all types of abnormalities. Nevertheless, KBrO3 at two doses was significantly increased in most types of abnormalities (P≤0.05) compared with the vehicle control. It was also observed that treatment with BV after injection with KBrO3 significantly decreased (P≤0.05) the most common types of abnormalities for both low and high doses comparing with that injected KBrO3 only. Meanwhile, sperm count significantly decreased (P≤0.05) in KBrO3-treated mice as compared with control. In contrast, mice injected with KBrO3 and supplemented with BV encountered significantly increased percentage of sperm count compared with KBrO3-treated mice ([Figure 3]).
|Table 2 Effect of potassium bromate and bee venom on sperm morphology of mice epididymis|
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|Figure 3 Effect of potassium bromate and bee venom on the sperm count of mice. Values with different letters are significantly different at P value less than or equal to 0.05.|
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[Figure 4] represents the DNA fragmentation patterns in the liver of the control and treated groups of mice. The results demonstrated that KBrO3 induced a ladder-like pattern in low and high dose of KBrO3 (lanes 3 and 4) compared with the control and BV groups (lanes 1 and 2), while the group treated with BV after injection with KBrO3 showed less smearing of DNA close to the control group (lanes 5 and 6).
|Figure 4 Agarose gel electrophoresis of DNA fragmentation. M, marker (100 bp); lane 1, control; lane 2, bee venom; lane 3, potassium bromate (KBrO3) (low dose); lane 4, potassium bromate (high dose); lane 5, bee venom+low-dose KBrO3, lane 6, bee venom+high-dose KBrO3.|
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Determination of catalase activity and malondialdehyde
Mice treated with KBrO3 at low and high doses showed significant decrease of CAT activity, while MDA values were significantly increased (P≤0.05) when compared with the untreated groups ([Table 3]). In contrast, treatment with BV after KBrO3 induced significant increase in CAT activity. Conversely, the activity of MDA significantly decreased (P≤0.05) for both low-dose and high-dose groups compared with that injected with KBrO3 only.
|Table 3 Effect of potassium bromate and bee venom on plasma malondialdehyde and catalase in mice|
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Determination of kidney function and protein content
The level of urea and creatinine of KBrO3-treated group (low and high doses) was significantly increased (P≤0.05) when compared with the control group ([Table 4]). In contrast, there was significant decrease in protein content as compared with the control. Treatment with BV after KBrO3 injection caused significant decrease of urea and creatinine levels and significant increase of protein content compared with that of KBrO3-treated animals.
|Table 4 Effect of potassium bromate and bee venom on protein and kidney function tests in serum of mice|
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Determination of liver function
[Table 5] represents the effect of BV and KBrO3 on liver function. Significant elevation (P≤0.05) in liver function marker (glutamic oxaloacetic transaminase, glutamic pyruvate transaminase, bilirubin, and alkaline phosphatase) levels was observed in mice treated with low and high doses of KBrO3 compared with the control group. However, mice treated with BV after low and high dose showed a significant decrease in liver function marker levels when compared with that injected with KBrO3 only.
|Table 5 Effect of potassium bromate and bee venom on liver function tests in serum of mice|
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| Discussion|| |
Previous reports have confirmed that KBrO3 can induce multiple organ toxicity mainly in the kidney and carcinogenic and, mutagenic effects in human and experimental animals ,. Animal and human studies have shown that BV may protect against oxidative stress and it is not genotoxic and do not produce oxidative damage at low concentrations .
According to genetic analysis, KBrO3 induced significant increases in frequencies of chromosomal aberrations and DNA fragmentation, which is similar to the results reported by Speit et al. , Kaya and Topaktaş  where they found that KBrO3 increased the number of chromosomal aberrations and DNA strand breaks. Moreover, KBrO3 in drinking water increased the number of aberrant metaphase cells in rat bone marrow . The oxidative stress induced by bromate has been postulated to play a role in its genotoxcicty through the lysosome damage mechanism that precedes oxidative DNA damage .
Regarding our results of micronucleus test, there was a marked increase in the number of micronuclei in high doses of KBrO3 than low doses which is in accordance with the study of Awogi et al.  where it has been reported that bromate increased the incidence of micronuclei, a sign of DNA damage, in the blood of MS/Ae and CD-1 mice strains. Also bromate elevated the frequency of cells with micronuclei in V79 Chinese hamsters . Similarly, the number of micronuclei was significantly increased in F344 rats intraperitoneally injected with bromate . Moreover, we have investigated the effect of KBrO3 on sperm morphology and the results showed that KBrO3 significantly increased the different types of sperm abnormalities. Our results is in coincidence with a previous study by Adil et al.  who found that the exposure of prepubertal rats to KBrO3 delay their growth, causes testicular hypoplasia, and deteriorate spermatogenesis, which lead to sterility in the future. A significant reduction was detected in the testes weight of rats fed with KBrO3 . Another previous study has shown significant decrease in epididymal sperm density in Sprague–Dawley male rats treated with bromate .
The biochemical analysis has shown that due to the oxidative stress exerted by the administration of KBrO3, a significant decrease of CAT activity and significant increase in MDA were recorded which is in line with the previous work of Edwards and Fuller . Other study reported that the mice group treated with bromate showed high levels of MAD and low levels of CAT . Parallel to these, in male rats treated with bromate, a decrease in the activity of CAT enzyme was recorded .
In addition, KBrO3 affected the kidney and liver functions, which are inconsistent with a former study carried out by Omer et al. , whereas treatment with KBrO3 caused a reduction in the total protein in Wistar albino rats. Mice treated with KBrO3 showed an increase in the urea and creatinine level and a decrease in protein level . Elevated levels of creatinine in the plasma confirmed that KBrO3 intake causes severe kidney damage . The hepatotoxic effect of KBrO3 referred to the reduction in an important antioxidant molecule glutathione, which can be used by many organs, including kidney and liver to resist the induced oxidative stress .
In contrast, the result of the present study shows that the intake of BV after KBrO3 recorded inhibitory effects against genotoxicity induced by KBrO3. This result was confirmed by several studies, which recorded the antiapoptotic effect of BV against the apoptosis induced by actinomycin D in normal hepatocyte cells . Treatment with BV combined with bleomycin reduced DNA fragmentation compared with bleomycin alone . Lee et al.  stated that the potentially reduced genotoxicity of BV in normal cells is due to its high reactivity against cell membrane which results in cell killing without reaching the DNA and exerting genotoxic effects and also it activates the forkhead subfamily transcription factors (FKHR and FKHR1), which have the ability to repair damaged DNA. In addition, a significant increase in hepatic GSH content following the administration of BV could be explained by the increased hepatic nitric oxide which protects cells from oxidative stress .
Regarding biochemical study, the results have shown that BV had significantly decreased the elevation of liver biomarkers, representing the hepato-protective effect of BV, which is in agreement with the results recorded by El-Bassiony et al.  and Merit , where they found that the injection of BV at different concentrations decreased the levels of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase. The hepato-protective effect of BV is exerted by inhibiting the secretion of proinflammatory cytokines, and decreasing the elevated serum aminotransferase enzymes injury .
Moreover, the present study demonstrated that kidney function markers, including urea and creatinine, were decreased following BV treatment. BV has protective effects on renal tubular injury (epithelial necrosis) . Treatment with BV significantly increased serum protein and albumin levels . Merit  recorded that injection with BV at different concentrations reduced the levels of serum parameters (albumin, globulin, and urea) of carcinogenic rats.
In addition, the treatment with BV in this study significantly decreased the level of MDA associated with increased CAT level in liver tissue which is similar to the results recorded by Abdel-Rahman et al. . This is attributed to the potent antioxidant property of BV that leads to a decrease in the levels of reactive oxygen species, which appears with the observations of BV affecting superoxide dismutase, glutathione, and CAT .
| Conclusion|| |
The results of the genetic and biochemical analysis revealed that BV has a potent ameliorating effect against high doses of KBrO3. This observation indicated the BV could be a potential therapeutic agent in the treatment of toxic effects of KBrO3. Further studies regarding the therapeutic effect of BV on KBrO3 toxicity in other animals and other organs are needed.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]