Bee venom genotoxicity on Saccharomyces cerevisiae cells - The role of mitochondria and YAP1 transcription factor.

The present work aims to clarify the genotype differences of a model organism Saccharomyces cerevisiae in response to bee venom. The study evaluated various endpoints including cell survival, induction of physiologically active superoxide anions, mitotic gene conversion, mitotic crossing-over, reverse mutations, DNA double-strand breaks, and Ty1 retrotransposition. The role of the intact mitochondria and the YAP1 transcription factor was also evaluated. Our results indicate a genotype-specific response. The first experimental evidence has been provided that bee venom induces physiologically active superoxide anions and DNA double-strand breaks in S. cerevisiae. The lack of oxidative phosphorylation due to disrupted or missing mitochondrial DNA reduces but not diminishes the cytotoxicity of bee venom. The possible modes of action could be considered direct damage to membranes (cytotoxic effect) and indirect damage to DNA through oxidative stress (genotoxic effect). YAP1 transcription factor was not found to be directly involved in cell defense against bee venom treatment.

Fatal Africanized bee attack in captive non-human primates (Saimiri ustus and Sapajus libidinosus).

Bee stings (BS) are a life-threatening issue and a growing concern for public health and animals in the Americas. We describe the clinical, pathological, and ultrastructural findings of a massive lethal bee attack in two non-human primates (NHPs). Both animals showed BS scattered throughout the skin, surrounded by a local reaction, diffuse pulmonary congestion, edema, hemorrhage, and remarkable degeneration and necrosis of renal epithelial cells from the proximal and distal tubules, characterizing a systemic bee envenomation reaction.

Bee Venom Causes Oxidative Stress, Biochemical and Histopathological Changes in the Kidney of Mice.

Accidents with venomous bees are a serious worldwide health concern. Since the kidney has been reported as the main venom-target organ, the present study was undertaken to investigate the in vivo nephrotoxic effect of Algerian bee venom (ABV) (Apis mellifera intermissa) collected in the middle east of Algeria. A preliminary study was performed on ABV to identify the ABV using SDS-PAGE analysis and to determine the in vivo intraperitoneal median lethal dose (LD50) using the Probit analysis test. In vivo nephrotoxic effect was assessed through the determination of physiological and kidney biochemical markers in mice intraperitoneally injected with ABV at doses of 0.76 (D1); 1.14 (D2) and 2.29 mg/kg body weight (bwt) (D3), corresponding respectively to LD50/15, LD50/10, and LD50/5 (i.p. LD50=11.48 mg/kg bwt) for seven consecutive days. Results revealed a marked decrease in body weight gain and food intake, and an increase in absolute and relative kidney weights in ABV D2 and D3 treated mice compared with controls. Furthermore, ABV D2 and D3 resulted in a significant increase in serum creatinine, urea, and uric acid. ABV-induced oxidative stress was evidenced by a significant increase in kidney MDA level, and a significant depletion in kidney GSH level, and catalase activity. Meanwhile, no marked changes in the above-mentioned parameters were noticed in ABV D1. Accordingly, the adverse nephrotoxic effect of ABV was proved by the dose-dependent kidney histological changes. In summary, the results of the present study evidence that ABV at doses of 1.14 (D2) and 2.28 mg/kg body weight (bwt) can cause marked changes in kidney biochemical and major antioxidant markers, and histological architecture.

Bee venom (Apis mellifera L.) rescues zinc oxide nanoparticles induced neurobehavioral and neurotoxic impact via controlling neurofilament and GAP-43 in rat brain.

This study investigated the possible beneficial role of the bee venom (BV, Apis mellifera L.) against zinc oxide nanoparticles (ZNPs)-induced neurobehavioral and neurotoxic impacts in rats. Fifty male Sprague Dawley rats were alienated into five groups. Three groups were intraperitoneally injected distilled water (C 28D group), ZNPs (100 mg/kg b.wt) (ZNPs group), or ZNPs (100 mg/kg.wt) and BV (1 mg/ kg.bwt) (ZNPs + BV group) for 28 days. One group was intraperitoneally injected with 1 mL of distilled water for 56 days (C 56D group). The last group was intraperitoneally injected with ZNPs for 28 days, then BV for another 28 days at the same earlier doses and duration (ZNPs/BV group). Depression, anxiety, locomotor activity, spatial learning, and memory were evaluated using the forced swimming test, elevated plus maze, open field test, and Morris water maze test, respectively. The brain contents of dopamine, serotonin, total antioxidant capacity (TAC), malondialdehyde (MDA), and Zn were estimated. The histopathological changes and immunoexpressions of neurofilament and GAP-43 protein in the brain tissues were followed. The results displayed that BV significantly decreased the ZNPs-induced depression, anxiety, memory impairment, and spatial learning disorders. Moreover, the ZNPs-induced increment in serotonin and dopamine levels and Zn content was significantly suppressed by BV. Besides, BV significantly restored the depleted TAC but minimized the augmented MDA brain content associated with ZNPs exposure. Likewise, the neurodegenerative changes induced by ZNPs were significantly abolished by BV. Also, the increased neurofilament and GAP-43 immunoexpression due to ZNPs exposure were alleviated with BV. Of note, BV achieved better results in the ZNPs + BV group than in the ZNPs/BV group. Conclusively, these results demonstrated that BV could be employed as a biologically effective therapy to mitigate the neurotoxic and neurobehavioral effects of ZNPs, particularly when used during ZNPs exposure.

Severe pathological changes in the blood and organs of SD rats stung by honeybees.

With the development of beekeeping, the risk of bee stings in humans is increasing. Severe and life-threatening toxic reactions can occur after multiple bee stings, and their pathogenesis has not been elucidated. To understand the effect of multiple bees (Apis mellifera) stings on the organism in a short period, we stung rats once and five times, respectively. Serum and organs were obtained after 3 h for analysis. The results indicated that skin erythema was more pronounced and hemolysis was more severe as the number of puncture wounds increased. After being stung by five bees, rats had dramatically higher serum levels of direct bilirubin, aspartate aminotransferase, creatine kinase and lactate dehydrogenase, producing more differential metabolites that affected mainly four metabolic pathways. In addition, the liver, kidney and heart showed significant congestion and inflammation. This study helps explain the organism's clinical response to bee venom and may be valuable in treating toxic reactions following bee stings.

Experimental envenomation with honeybee venom melittin and phospholipase A2 induced multiple ultrastructural changes in adrenocortical mitochondria.

Bee stings represent a public health subject, but the mechanisms involved in bee venom toxicity are not yet fully understood. To evaluate the reactions of adrenocortical cells, through which organisms respond to stress, two honeybee venom components: melittin (Mlt) and phospholipase A2 (PLA2) were tested as potential chemical stressors. Modifications were investigated with transmission electron microscopy and microanalysis. A single dose of Mlt (31 mg/kg) or PLA2 (9.3 mg/kg) was injected in rats of groups ML and PL; daily doses of Mlt (350 µg/kg) or PLA2 (105 µg/kg) were injected 30 days in rats of groups M30 and P30. Adrenocortical cells in ML group showed ultrastructural degenerative alterations of nuclei, endoplasmic reticulum, and mitochondria that exhibited lipid inclusions and mitochondrial cristae (MC) re-organized into mono- or multimembrane large vesicles, and whorls of membranes. Many MC were degenerated. In the M30 group, similar ultrastructural changes, but of lower amplitude were noted; lipid cytosolic droplets were heterogenous. MC diameters in Mlt groups (melittin treated groups) were significantly higher than in control (C) group. In PL group, mitochondria contained large lipid inclusions, vesicular MC of different sizes and multiple membranes, and debris, or whorl structures. In P30 group MC were tubular with increased diameters. In both PLA2 groups (PLA2 treated groups) MC were significantly larger than in C group. We concluded that Mlt and PLA2 were powerful stressors, toxic at the tested doses, cellular reactions concerning in all groups mainly mitochondria, but also other cellular compartments. Apart from degenerative regression of MC, the rearrangement of tubular MC occurred into one or multiple large multimembrane vesicular MC. Reactions to the high doses were more pronounced, with the highest amplitude in ML group, and the lowest in P30 group.

Natural and Induced Tolerance to Hymenoptera Venom: A Single Mechanism?

Inducing tolerance in Hymenoptera-allergic patients, bee venom immunotherapy (BVIT) is a widely accepted method to treat severe allergy to bee stings. In order to increase the existing knowledge on the underlying immunological mechanisms and look for possible biomarkers predictive of efficacy, a group of 20 bee-venom-allergic patients (AG) were thoroughly examined during their first year of BVIT. In addition, the results of treated patients with those of an untreated group of 20 tolerant beekeepers (TG) who had previously shown a firm suppressor-regulatory profile were compared. Tolerance in AG patients was invariably associated with a significant regulatory response characterised by the expansion of Helios- subpopulation and increased IL-10, specific IgG4 (sIgG4), and kynurenine levels. Although specific IgE (sIgE) levels increased transiently, surprisingly, the T helper type 2 (Th2) population and IL-4 levels rose significantly after one year of immunotherapy. Thus, the picture of two parallel phenomena emerges: a tolerogenic response and an allergenic one. Comparing these results with those obtained from the TG, different immunological mechanisms appear to govern natural and acquired tolerance to immunotherapy. Of particular interest, the kynurenine levels and T regulatory (Treg) Helios- population could be proposed as new biomarkers of response to BVIT.

Anticancer Activity of Bee Venom Components against Breast Cancer.

While the survival rate has increased due to treatments for breast cancer, the quality of life has decreased because of the side effects of chemotherapy. Various toxins are being developed as alternative breast cancer treatments, and bee venom is drawing attention as one of them. We analyzed the effect of bee venom and its components on breast cancer cells and reviewed the mechanism underlying the anticancer effects of bee venom. Data up to March 2022 were searched from PubMed, EMBASE, OASIS, KISS, and Science Direct online databases, and studies that met the inclusion criteria were reviewed. Among 612 studies, 11 were selected for this research. Diverse drugs were administered, including crude bee venom, melittin, phospholipase A2, and their complexes. All drugs reduced the number of breast cancer cells in proportion to the dose and time. The mechanisms of anticancer effects included cytotoxicity, apoptosis, cell targeting, gene expression regulation, and cell lysis. Summarily, bee venom and its components exert anticancer effects on human breast cancer cells. Depending on the mechanisms of anticancer effects, side effects are expected to be reduced by using various vehicles. Bee venom and its components have the potential to prevent and treat breast cancer in the future.

Incidence Rate of Bee Venom Acupuncture Related Anaphylaxis: A Systematic Review.

BACKGROUND: Bee venom acupuncture (BVA) is an effective treatment method for various diseases. Bee venom, however, can cause adverse effects, even rarely including life-threatening anaphylaxis, so safety-related evidence is required. In this study, we systematically estimated the incidence rate of anaphylaxis in response to BVA. METHODS: We searched eight databases (MEDLINE (Pubmed), EMBASE, Cochrane Central Register of Controlled, KISS, KMBASE, Koreamed, OASIS, and NDSL) and systematically reviewed the articles that met the inclusion/exclusion criteria. RESULTS: Among 225 potentially relevant articles, 49 were selected for this study. The overall incidence rate of anaphylaxis in response to BVA was 0.045% (95% CI 0.028-0.062). Women (0.083%, 95% CI 0.010-0.157) showed a higher incidence rate than men (0.019%, 95% CI -0.018 to 0.055), while the incidence for patients who had a skin test conducted (0.041%, 95% CI 0.011-0.072) was not significantly different compared to that obtained for patients for which there was no information about a skin test (0.047%, 95% CI 0.026-0.067). The publication year affected the incidence rate: it was highest before 1999 (1.099%, 95% CI -1.043 to 3.241), lower between 2000 and 2009 (0.049%, 95% CI 0.025-0.073), and lowest between 2010 and 2021 (0.037% 95% CI 0.014-0.060). CONCLUSIONS: In this study, we provide reference data about risk size and factors of BVA-related anaphylaxis, which is essentially required for BVA application in clinics.

Anaphylactic shock with pulmonary eosinophilic infiltration due to honeybee attack in a donkey: case report / [Choque anafilático com infiltração eosinófila pulmonar devido a ataque de abelhas em um burro: relato de caso]

A case of a donkey attacked by Africanized honeybee is reported here with clinical signs of agitation, dehydration, congestion of the ocular mucous membranes, tongue edema, tachycardia and inspiratory dyspnea, and progression to death. At necropsy, diffuse, severe subcutaneous edema at face and cervical regions and severe diffuse pulmonary hyperemia with abundant edema without parenchymal collapse were observed. Microscopically, marked, diffuse deep dermis and panniculus carnosus edema and marked diffuse alveolar edema, with moderate population of eosinophils predominantly around larger caliber vessels were noted. The final diagnosis of anaphylactic shock was supported by history, clinical signs, and anatomic pathology findings. This is the first report of a honeybee attack with pulmonary eosinophilic infiltration in a mammal.(AU)

Descreve-se um caso de ataque de abelha africanizada em um burro, com sinais clínicos de agitação, desidratação, mucosas oculares congestas, edema de língua, taquicardia e dispneia inspiratória, com progressão e morte. Na necropsia, foram verificados edema subcutâneo difuso grave nas regiões de face e cervical, hiperemia pulmonar difusa grave com edema abundante e sem colapso do parênquima. Microscopicamente, foram observados edema marcado difuso na derme profunda e panículo carnoso e edema alveolar difuso acentuado, com população moderada de eosinófilos predominantemente em torno de vasos de maior calibre. O diagnóstico de choque anafilático foi baseado no histórico, em sinais clínicos e em achados anatomopatológicos. Este é o primeiro relato de ataque de abelhas com infiltração eosinofílica pulmonar em um mamífero.(AU)

CLINICAL OBSERVATIONS OF BEE ENVENOMATION IN TOUCANS.

There are numerous reports on envenomation, even fatal, secondary to bee attacks in humans and other mammals. In birds, reports on those incidents are scarce and there are none regarding honeybee (Apis mellifera) stings in toucans. In the first case presented, an adult female red-breasted toucan (Ramphastos dicolorus) received at least five bee stings in the periophthalmic area. Within 5 h the bird was lethargic and dehydrated. The urates were yellowish. Three days later the bird showed a moderate anemia, but no changes in the leukocyte count, beyond an elevated heterophil: lymphocyte ratio. Blood chemistry showed hyperglycemia, hypoalbuminemia and elevated aspartate aminotransferase and creatine kinase. Alterations in electrolyte values were also noted. Fourteen days later bile acid elevation was observed. Hematocrit levels normalized after 2 wk. A second incident involved a breeding pair of toco toucans (Ramphastos toco). While the female toco toucan received 10 stings and showed mild clinical manifestations, the male toco toucan was more severely attacked, receiving 40 stings, and died overnight. Despite the relative gravity of the attack (in terms of number of stingers in relation to body weight) both surviving birds recovered in less than 2 wk. To the authors' knowledge, fatal bee envenomation in birds has been reported only in pigeons and macaws. The findings described in this report suggest that toucans are less sensitive to bee venom when compared with pigeons and may have higher tolerance to bee venom compared with mammals. Honeybee envenomation must be considered a potential threat when considering toucan husbandry in zoos and collections.

Case Report: Acute Kidney Failure due to Massive Envenomation of a Two-Year-Old Child Caused by Killer Bee Stings.

A hybrid species of Brazilian bee has proliferated on the South American continent since 1956. We describe a "killer bee" swarm attack on a 2-year-old girl in French Guiana. The patient weighed 10 kg, and approximately hundreds of bees' stingers were removed, that is, 10 stings/kg. Our patient survived without long-term sequelae. The management of her condition required admission into intensive care for renal failure due to acute tubular necrosis and severe rhabdomyolysis. We emphasize the importance of early medical intervention, clinical surveillance, and biological monitoring at the hospital to prevent a toxic chain reaction that could prove fatal within 72 hours.

PGD2 /CRTH2 signaling promotes acquired immunity against bee venom by enhancing IgE production.

IgE-dependent/independent activation of mast cell (MC) has been assumed to play a host defensive role against venom injection in skin. However, its detailed mechanisms remain unknown. We aimed to investigate the contribution of MC-derived prostaglandin D2 (PGD2 )-mediated signaling in host defense against bee venom (BV). To achieve this, we utilized gene-deficient mice of a PGD2 receptor, chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). We first confirmed that subcutaneous injection of BV produced PGD2 equally in wild-type (WT) and CRTH2-deficient (Crth2-/- ) mice skins. The BV injection dropped body temperature and impaired kidney equally in both lines of mice. In WT mice, pre-injection of BV (3 weeks) significantly inhibited the hypothermia and kidney impairment caused by second BV injection. In contrast, this pre-injection was not effective for the second BV injection in Crth2-/- mice. We also found that BV injections increased serum BV-specific IgE levels in WT mice, and its serum transfused mice improved the BV-induced hypothermia in naïve WT mice. In contrast, serum BV-specific IgE level was significantly lower in Crth2-/- mice. FACS analysis showed the BV injection stimulate migration of dendritic cells (DCs) into regional lymph nodes in WT mice. In Crth2-/- mice, its number was significantly smaller than that of WT mice. In conclusion, PGD2 /CRTH2 signaling plays defensive role against second BV injection. This signaling promotes BV-specific IgE production at least partially by promoting DCs migration into regional lymph node.

Lipolytic Activity of a Carboxylesterase from Bumblebee (Bombus ignitus) Venom.

Bee venom is a complex mixture composed of peptides, proteins with enzymatic properties, and low-molecular-weight compounds. Although the carboxylesterase in bee venom has been identified as an allergen, the enzyme's role as a venom component has not been previously elucidated. Here, we show the lipolytic activity of a bumblebee (Bombus ignitus) venom carboxylesterase (BivCaE). The presence of BivCaE in the venom secreted by B. ignitus worker bees was confirmed using an anti-BivCaE antibody raised against a recombinant BivCaE protein produced in baculovirus-infected insect cells. The enzymatic activity of the recombinant BivCaE protein was optimal at 40 °C and pH 8.5. Recombinant BivCaE protein degrades triglycerides and exhibits high lipolytic activity toward long-chain triglycerides, defining the role of BivCaE as a lipolytic agent. Bee venom phospholipase A2 binds to mammalian cells and induces apoptosis, whereas BivCaE does not affect mammalian cells. Collectively, our data demonstrate that BivCaE functions as a lipolytic agent in bee venom, suggesting that BivCaE will be involved in distributing the venom via degradation of blood triglycerides.

Beekeepers who tolerate bee stings are not protected against SARS-CoV-2 infections.

A survey on 5115 beekeepers and 121 patients treated with bee venom by an apitherapy clinic in the Hubei province, the epicenter of COVID-19 in China, reported that none of the beekeepers developed symptoms associated with COVID-19, the new and devastating pandemic. The hypothesis that immunity to bee venom could have a preventive effect was expressed and the authors of the Chinese survey suggested that the next step should be animal experiments on monkeys. We believed that before starting such studies, a second independent survey should verify the findings and define the hypothesis more clearly. Thus we asked all German beekeepers to complete an assessment form which would summarize their experiences with COVID-19. In contrast to the Chinese study we found that two beekeepers had died from a SARS-CoV-2 infection and forty-five were affected. The reaction to bee stings (none; mild swelling; severe swelling) correlated with the perceived severity of the SARS-CoV-2-infection-associated symptoms - exhaustion and sore throat. Beekeepers comorbidity correlated with problems with breathing at rest, fever, and diarrhea. Our results did not confirm the findings of the Chinese study. However, since the antiviral effects of bee venom have been found in several studies, we cannot exclude that there could be a direct preventive or alleviating effect when bee venom is administered during the infection.

Massive Africanized honeybee stings in two hair sheep and a mare.

The findings of massive Africanized honeybee stings in two hair sheep and a mare are reported. One sheep died 15 h after attack, and the survivors developed skin necrosis on the sting sites. Pathological evaluation revealed necrosis in the dermis, degeneration of the tubular epithelial cells, and multifocal hemorrhages in heart and spleen. The massive attack by Africanized honeybees induced lesions in the skin, heart, muscles, kidneys, and lungs.

Factor XII-Deficient Chicken Plasma as a Useful Target for Screening of Pro- and Anticoagulant Animal Venom Toxins.

The sensitivity of vertebrate citrated plasma to pro- and anticoagulant venom or toxins occurs on a microscale level (micrograms). Although it improves responses to agonists, recalcification triggers a relatively fast thrombin formation process in mammalian plasma. As it has a natural factor XII deficiency, the recalcification time (RT) of chicken plasma (CP) is comparatively long [≥ 1800 seconds (s)]. Our objective was to compare the ability of bee venom phospholipase A2 (bvPLA2) to neutralize clot formation induced by an activator of coagulation (the aPTT clot) in recalcified human and chicken plasmas, through rotational thromboelastometry. The strategy used in this study was to find doses of bvPLA2 that were sufficient enough to prolong the clotting time (CT) of these activated plasmas to values within their normal RT range. The CT of CP was prolonged in a dose-dependent manner by bvPLA2, with 17 ± 2.8 ng (n = 6) being sufficient to displace the CT values of the activated samples to ≥ 1800 s. Only amounts up to 380 ± 41 ng (n = 6) of bvPLA2 induced the same effect in activated human plasma samples. In conclusion, the high sensitivity of CP to agonists and rotational thromboelastometry could be useful. For example, during screening procedures for assaying the effects of toxins in several stages of the coagulation pathway, such as clot initiation, formation, stability, strength, or dissolution.

Bee venom attenuates Porphyromonas gingivalis and RANKL-induced bone resorption with osteoclastogenic differentiation.

Porphyromonas gingivalis (P. gingivalis) is one of the major periodontal pathogens leading to inflammation and alveolar bone resorption. Bone resorption is induced by osteoclasts, which are multinucleated giant cells. Osteoclastic bone resorption is mediated by enhanced receptor activator of nuclear factor-kappa B ligand (RANKL) signaling. Therefore, the down-regulation of RANKL downstream signals is regarded as an effective therapeutic target in the treatment of bone loss-associated disorders. The aim of this study was to evaluate whether purified bee venom (BV) could attenuate P. gingivalis-induced inflammatory periodontitis and RANKL-induced osteoclast differentiation. Inflammatory periodontitis induced by P. gingivalis increased alveolar bone resorption and increased expression of TNF-α and IL-1ß, while BV treatment resulted in decreased bone loss and pro-inflammatory cytokines. Similarly, RANKL-induced multinucleated osteoclast differentiation and osteoclast-specific gene expression, such as nuclear factor of activated T cells 1 (NFATc1), cathepsin K, tartrate-resistant acid phosphatase (TRAP), and integrin αvß3 were significantly suppressed by treatment with BV. We show that BV reduces P. gingivalis-induced inflammatory bone loss-related periodontitis in vivo and RANKL-induced osteoclast differentiation, activation, and function in vitro. These results suggest that BV exerts positive effects on inflammatory periodontitis associated osteoclastogenesis.

A biomimetic nanoparticle-enabled toxoid vaccine against melittin.

BACKGROUND: Melittin, the main active peptide ingredient of bee venom, can cause severe cell membrane lysis due to its robust interaction with negatively charged phospholipids. So far, no effective anti-melittin vaccine has been developed to protect people from undesired melittin intoxication. METHODS: Herein, we prepared a polydiacetylene (PDA) nanoparticle with cell membrane-mimic surface to complex melittin, forming an anti-melittin vaccine (PDA-melittin). RESULTS: PDA nanoparticles could effectively combine with melittin and neutralize its toxicity. PDA-melittin nanocomplex is demonstrated to enhance melittin uptake by DCs and stimulate strong melittin-specific immunity. Mice immunized with PDA-melittin nanocomplex showed higher survival rate after exposion to melittin than untreated mice. CONCLUSION: The PDA-melittin nanocomplex can efficiently and safely generate a specific immunity against melittin to protect body from melittin intoxication, providing a new method with potential clinical application for the treatment of melittin intoxication.