RADIOPROTECTION: MECHANISMS AND RADIOPROTECTIVE AGENTS INCLUDING HONEYBEE VENOM

Review article

RADIOPROTECTION: MECHANISMS AND RADIOPROTECTIVE AGENTS INCLUDING HONEYBEE VENOM

E. A. VARANDA

, D. C. TAVARES

1 Department of Biological Sciences, School of Pharmaceutical Sciences of Araraquara, São Paulo State University, State of São Paulo, Araraquara, SP, Brazil, 2 Center for the Study of Venoms and Venomous Animals, CEVAP, UNESP, São Paulo State University, Botucatu, SP, Brazil.

ABSTRACT. Since 1949, a great deal of research has been carried out on the radioprotective action of chemical substances. These substances have shown to reduce mortality when administered to animals prior to exposure to a lethal dose of radiation. This fact is of considerable importance since it permits reduction of radiation-induced damage and provides prophylactic treatment for the damaging effects produced by radiotherapy. The following radioprotection mechanisms were proposed: free radical scavenger, repair by hydrogen donation to target molecules, formation of mixed disulfides, delay of cellular division and induction of hypoxia in the tissues. Radioprotective agents have been divided into four major groups: the thiol compounds, other sulfur compounds, pharmacological agents (anesthetic drugs, analgesics, tranquilizers, etc.) and other radioprotective agents (WR-1065, WR-2721, vitamins C and E, glutathione, etc.). Several studies revealed the radioprotective action of Apis mellifera honeybee venom as well as that of its components mellitin and histamine. Radioprotective activity of bee venom involves mainly the stimulation of the hematopoietic system. In addition, release of histamine and reduction in oxygen tension also contribute to the radioprotective action of bee venom.

KEY WORDS: radiation , protective agents, honeybee venom.

INTRODUCTION

Ionizing radiation is any electromagnetic wave or particle capable of producing ions in its passage through the matter, causing immediate chemical alterations in biological tissues. These alterations produce a metabolic disarrangement which after days or weeks can lead either to cell damage or ultimately to cell or organism death. Ionizing radiation damage is caused by either a direct interaction with target molecules or indirectly by formation of chemically and pharmacologically active elements produced mainly by water molecules. Radiation absorbed interacts almost exclusively with electrons of atoms and tissue molecules. Although this process forms positive ions, the decisive factor on the biological viewpoint, is that these molecules contain an unpaired electron in one of its orbitals, forming free radicals. Free radicals are molecules that exist only for a fraction of seconds. Due to predominance of water in the tissues, most of the ionizing events occur in this molecule. Water radiolysis generates molecules of hydrogen peroxide (H2O2), molecular hydrogen (H2) and a number of highly active radicals such as hydrogen radical (H ), hydroxyl radical (OH ), hydroperoxyl radical (HO ) and superoxide (O2 ). Sulfhydryl compounds are highly sensitive to indirect action of radiation. Disulfide compounds and sulfur atoms of complex organic molecules also are highly susceptible to the attack of the above-mentioned radicals. Chemical alterations of nucleic acids such as breaks of hydrogen bonds, break of base-sugar binding, sugar oxidation, break of nucleotide strand and release of terminal phosphates are also caused by reactions of free radicals(53).

A new line of research appeared after the discovery that animals could be partially protected against deleterious effects of ionizing radiation by administration of certain specific chemical compounds.

Since 1949, a great deal of research has been conducted on the radioprotective action of chemical substances which reduce mortality if administered to mammals prior to exposure to a lethal dose of radiation. This fact is of considerable importance since it permits reduction of radiation-induced damage as well as provides prophylactic treatment for damaging effects caused by of radiotherapy.

Studies of Patt(49) and Bacq et al.(6) demonstrated the radioprotective effect of aminothiols against lethality in a large number of animal species. These studies created hope that these compounds could be used in humans. Conventional radiotherapy is limited by tolerance of normal tissue. Thus, a compound which can protect normal tissue and also permit an increase in the radiation dose used has been searched for. The relation between dose and cure of a number of tumors indicate that slight increases in the tolerated doses might bring about a significant enhancement in the rate of local control. In this way, any compound that could protect normal tissues, and at the same time destroy malignant tumors, could be of considerable benefit.

RADIOPROTECTION MECHANISMS

A considerable number of investigations testing compounds in several biological systems were carried out. Thus, the following radioprotection mechanisms were proposed: free radical scavenger, repair by hydrogen donation to target molecules, formation of mixed sulfides, delay of cellular division and induction of hypoxia in the tissues.

The mechanism of free radical scavenger suggests that certain agents are oxidized by free radicals, forming stable compounds incapable of reacting with other cellular components. This mechanism prevents the free radicals from reacting with the cell vital components.

Another mechanism which has been demonstrated with polymers is the repair by hydrogen donation. If a R-H molecule is converted into a R (radical R ) by exposure to radiation, a protective agent can donate a hydrogen atom to this radical, restoring it to its original state(10).

The formation of mixed disulfides is a mechanism proposed for aminothiols and involves radioprotector binding to cellular components. Sulfhydryl compounds of the aminothiols form mixed disulfides with sulfhydryl compounds of cellular proteins. When one of these disulfides is attacked by free radicals, one of the sulfur atoms is reduced and the other is oxidized. If the sulfur atom of the protein is reduced and the sulfur atom of the protective agent is oxidized, the protein is not damaged. Therefore, the cellular proteins are protected in 50% of the cases. This theory considers oxidation of sulfhydryl compounds of cellular proteins as the main factor for occurrence of radiation-induced damage.

Binding of aminothiols and disulfides to DNA has been considered a potentially important factor in radioprotection. In view of these considerations, Brown(12) proposed that the sulfhydryl compounds of the radioprotective aminothiols act by binding to DNA, and thereby, reversibly inhibit replication and stabilize their structure, which provides additional time for repair.

Oxidation of the thiols consumes enough oxygen to reduce its tension, and it has already been demonstrated that hypoxia is radioprotective. In addition, it seems that induction of hypoxia may, in certain conditions, be a contribution of the thiols to radioprotection. However, other mechanisms might be involved, since some compounds exert radioprotective activity without altering oxygen tension on the tissues. There is evidence of the existence of more than one radioprotective mechanism of a certain compound, and that one of the compounds might be more or less important, depending on the irradiated system and on the specific radiation conditions(56).

Other radioprotective mechanisms have been reported, although little is known about them. One of these mechanisms is protection by substitution in which the protective agents work as spare parts being capable of substituting cellular constituents if the biochemical functions were irreversibly damaged(7,42). Other mechanisms are: protection by alteration of cellular metabolism, stress caused by irradiation or by other means that leads to an enhancement of radioresistance, and castration which increases radioresistance in male rats and decreases it in female rats (9,42).

Modification of ionizing radiation-induced damage in mammals has been extensively studied either from the therapeutic or the prophylactic viewpoint. Experimentally, the most successful therapy is transplantation of hematopoietic tissue after irradiation(60,72), whereas prophylaxis can be, made by the administration of protective agents prior to irradiation(5). These agents are either chemical protectors, such as cysteine and 2-aminoethylisothio-uronium bromide hydrobromide (AET), which reduce radiation-induced damage, or biostimulant protectors, such as estrogen and endotoxin, which enhance regeneration of hematopoietic tissue having also been considered as radioprotective mechanisms.

RADIOPROTECTIVE AGENTS

The terms radioprotection and radioprotective agents were introduced by Dale(18) who carried out studies using enzymes as indicative molecules. However, the first study that aroused interest about radioprotective drugs for humans was conducted by Patt et al.(50). In this study, cysteine, a sulfur-containing amino acid was administered intravenously to rats 15 minutes prior to receiving a lethal dose of radiation. A significant increase of surviving animals was observed. With passing time, more efficient radioprotective agents were found, and they generally involved alterations in the chemical structure of sulfur-containing compounds. In 1957, according to Delaney et al.(19), the USA Army started a program to develop radioprotective drugs as well as studies about functions of natural compounds and of enzymatic systems in the mediation of radiation-induced damage.

According to Prasad(56), the radioprotective agents have been divided into four major groups, as follows:

THIOLS: The thiols include cysteine, cysteamine, cystamine, AET and 2-mercaptoethylguanidine (MEG). The sulfhydrylamines are potent agents which reduce temperatures and physiological pH. The dose reduction factor (DRF) of various compounds ranges from 1.4 to 2.0. This class of compounds is characterized by the sulfhydryl compounds (SH) and amine (NH2) separated by 2 carbon atoms.

OTHER COMPOUNDS WHICH CONTAIN THE -SH RADICAL: Hundreds of compounds showing the -SH radical have been tested, but, only the following showed to have radioprotective action: thiourea, thiouracil, dithiocarbamate, dithioxamides, thiazolines, sulfoxides and sulfones.

PHARMACOLOGICAL AGENTS:

Anesthetic drugs and alcohol: Drugs commonly used as anesthetics are not effective in radioprotection, while alcohol ingestion causes a respiratory depression and, consequently, tissue hypoxia, which can be one of the most important protective mechanisms against radiation.

Analgesics: Morphine and heroin increase the LD50 from 609 to 830 R (Roengten) in mice. Sodium salicylate enhances survival in 50% after treatment with 700 R.

Tranquilizers: Injection of reserpine in mice 12 h prior to radiation exposure increases LD50 from 605 to 825 R for males and from 635 to 727 R for females. However, reserpine is ineffective in rats.

Cholinergic drugs: Acetylcholine, metacholine (acetyl-ß-metilcholine and carbaminoylcholine have some protective effect in rats, but choline in itself is ineffective.

Epinephrine and norepinephrine: Epinephrine protects animals against radiation-induced death but norepinephrine does not. Equal doses of both drugs increase arterial blood pressure to the same extent, but norepinephrine decreases oxygen tension in spleen in 48% and epinephrine in 90%. Methoxamine increases LD50 from 825 to 1100 R (DRF=1.3) in rats.

Dopamine: Dopamine administered prior to whole body radiation exposure, protects 80% of irradiated mice with 700 R, a dose which usually causes 100% lethality.

Histamine: DRF values for histamine in mice of the strain CBA is approximately 1.5, while in mice of the strain C57 is 1.1. Oxygen tension in the spleen after treatment with histamine decreases from 77 to 93%. The decrease of O2 is believed to be the main protection mechanism.

Serotonin: Serotonin is as effective as cysteamine in mice. DRF value is approximately 1.84.

Hormones: DRF value of hormones such as adrenal hormones and thyroid hormones is approximately 1.1. Estrogen and colchicine also possess some radioprotective effect.

OTHER RADIOPROTECTIVE AGENTS: There is a variety of other radioprotective agents as follows: cyanide, derivatives of nucleic acids (e.g., ATP), sodium fluoracetate, para-aminopropiophenone (PAPP), mellitin, endotoxins, imidazole, adenosine 3',5'-cyclic monophosphate (cAMP), antibiotics, lipids (e.g. olive oil), erythropoietin, carbon monoxide (competes with hemoglobulin), hydrochloric mercaptoethylamine (MEA), serotonin, sodium hydrogen S-(2-aminoethyl) phosphorothioic acid (WR-638), S-2-(3-aminopropylamino)ethyl phosphorothioic acid (WR-2721) and S-2-(3-aminopropylamino) propylphosphorothioic acid (WR-44923).

Smith and Mackinley(61), analyzing the radioprotective effects of phenyldrazine (PhNHNH2), Horiuchi and Miyamoto(32), studying the effects of bestatin, a substance known to protect mice against cyclophosphamide-induced damage, and Real et al.(57), analyzing the effects of a protein associated with a polysaccharide called AM5 verified that all these substances exhibit radioprotective effects because they stimulate the hematopoietic proliferation.

The thiol compounds WR-2721 and 2-mercaptopropionylglycine are radioprotective chemicals which should be present in human's and animal's bodies during irradiation. These substances have attracted the attention of a number of researchers because since they were discovered, they have been protecting mammals from radiation-induced death. Most studies conducted examined the lethality of organisms, but little was learned about the protective effects of these compounds on genetic material(28).

According to Milas et al.(46), the main radioprotective mechanism of the aminothiol N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065) and of its phosphorilated derivative WR-2721 is their capacity of scavenging free radicals formed during exposure to ionizing radiation. In addition, the radioprotective effect of these agents is higher on normal tissues than on malignant tissues, revealing their great importance in cancer radiotherapy.

According to Littlefield and Hoffmann(43), the efficacy of WR-2721 depends on its transformation into its free thiol, WR-1065, which reduces radiation-induced genetic damage, including single and double DNA-strand breaks, chromosomal aberrations, micronuclei and hprt mutations in mammalian cells. The mechanisms by which WR-1065 confers radioprotection are complex and might be related to scavenging of hydroxyl radicals, hydrogen donation to DNA radicals (chemical repair) and still produces anoxia close to DNA.

Although the capacity of the thiols in the protection of mammalian cells against lethal effects caused by ionizing radiation is well established, a number of aspects of radioprotective mechanisms are not well understood yet. Certain endogenous substances might modulate the radioprotective capacity of the aminothiols. One of these substances is glutathione (GSH), a non-proteic thiol found in mammalian cells and essential to the maintenance of several biochemical processes dependent on reduction and oxidation reactions that could affect cell susceptibility to radiation-induced damage(55). High levels of GSH are present in the body and have several protective functions, presenting a radioprotective effect by scavenging of free radicals similar to that of cysteine and cysteamine(14).

In animal experiments, Winters(75) observed that the level of glutamine in plasma decreased significantly after irradiation in the abdomen. Additional studies revealed that glutamine supplementation in the diet before or after irradiation reduced some of the side effects associated with the gastrointestinal tract, accelerated healing of the intestinal mucosa, reduced morbidity, mortality and bacterial translocation in the lumen(39,40,41,66).

Tavares and Takahashi(70) observed that acute treatment of rats with glutamine reduced the frequency of gamma radiation-induced chromosomal aberrations, even though it is not statistically significant.

A possible mechanism by which glutamine exerts a radioprotective effect is by preventing free radical-induced damage produced by radiation. This may occur by processes still unknown or by the action of glutathione, of which glutamine is a precursor(31,75).

Sarma and Kesavan(58) reported that a number of micronutrients usually found in the diet are also known for modifying ionizing radiation-induced damage. These authors analyzed the effects of vitamins C and E on bone marrow chromosomes of mice exposed to 1 Gy of gamma radiation and verified that vitamins C and E reduced significantly the frequency of chromosomal aberrations. This radioprotective effect could be explained by scavenging of free radicals, since these vitamins are well-known antioxidants in vivo when present in biological systems during exposure to radiation.

El Nahas et al.(22), analyzing the frequency of chromosomal aberrations in bone marrow of rats observed a radioprotective effect of vitamin C, but no effect was observed with vitamin E. Hydrogen peroxide and hydroxyl radicals produced by radiation causing cellular damage are not scavenged by vitamin E, which is a singlet oxygen scavenger. However, Felemovicius et al.(25) demonstrated the radioprotective effect of vitamin E both in its water-soluble form (alpha-tocopherol phosphate) and in its fat-soluble form (alpha-tocopherol acetate) on the X-radiation-induced damage on intestinal mucosa in rats.

The most extensively studied cytokines regarding their radioprotective action are: interleukin-1 (IL-1), tumor necrosis factor alpha (TNF- ), granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF)(21,76). One of the possible mechanisms proposed to explain the radioprotective action of IL-1 is that it stimulates stem cells and hematopoiesis(47).

Immunomodulators are another class of radioprotectors that can enhance survival in irradiated animals. It has been suggested that these agents mediate their radioprotective effects by mechanisms, such as enhancement of the proportion of hematopoietic stem cells into more radioresistant phase of the cell cycle or increase of the size of the postirradiation stem cell population(1,13). Broncho-Vaxon, lyophilized fraction of bacterial extract (free of endotoxins) from eight strains, is one of the immunomodulators(54) with radioprotective activity(23,24). Radioprotective activity of the combined administration of WR-2721 and Broncho-Vaxon on the hematopoietic system and circulating blood of mice showed to be more effective than the individual administration of one of these immunomodulators(44).

The immunomodulator AS101, (ammonium trichloro(dioxyethylene-O-O') Tellurate) stimulates the production of a variety of cytokines(35,68) and presents radioprotective activity in mice(36). IL-1, IL-6, TNF- and the c-kit ligand (protoncogene bound to stem cell factor) demonstrate a major role of AS101 in radioprotection(37).

The natural polyamines putrescine (1,4-Diaminobutane), spermidine and spermine are present in all mammalian cells(69). Studies about molecular mechanisms accounting for the radioprotective role of polyamines started recently(30). It has been shown that polyamines protect against the radiation-induced loss of DNA transforming activity, mainly by scavenging of hydroxyl radicals. Spotheim-Maurizot et al.(67) reported the radioprotective effect of these polyamines on the radiolysis of pBR322 plasmid DNA. A very efficient protection was observed against single and double-strand break in the presence of spermine and spermidine, and a less efficient protection in the presence of putrescine.

Other radioprotectors have been studied, among them the nitroxide Tempol (4-hydroxy-2,2,6,6,-tetramethylpiperidine-1-oxyl), which provides protection against radiation-induced chromosomal aberrations, mainly double-strand breaks in human peripheral blood lymphocytes(34). Calcium antagonists (diltiazem, nifedipine and nimodipine) also exert radioprotective action against gamma radiation. These compounds inhibit calcium influx through plasma membrane, thereby, influencing several cellular functions(26).

Stobadine is a drug of pyridoindole structure with antioxidant properties and potential pharmacological use. This drug reduces significantly the frequency of gamma radiation-induced chromosomal damage in mice. Enhancement of the repair of DNA damage and the activity as scavenger of hydroxyl radicals produced by radiation are radioprotective mechanisms involving stobadine(15).

In addition to the already described radioprotective compounds, the effect of bacterial endotoxins has been known for over 30 years(45,62). Subsequent studies demonstrated that radioprotection can also be achieved using preparations of detoxified endotoxins. These studies suggested that the locus for radioprotection is located in a region of the endotoxin molecule different from the locus accounting for toxicity. Nowotny et al.(48) and Urbaschek(71) confirmed this hypothesis. The mechanism by which an endotoxin increases survival of irradiated animals is not yet well understood, but it is believed that this survival increase is associated with the capacity of stimulating the repair of the hematopoietic system(63,64). Similarly, Snyder et al.(65) tested the radioprotective action of monophosphoryl (detoxified) lipid A (LAM) isolated from endotoxin lipid A of Salmonella minnesota R 595. LAM produced a DRF value of 1.2.

RADIOPROTECTIVE ACTION OF HONEYBEE VENOM

Studies about the radioprotective action of venoms are scarce and basically restricted to bee venom and some of its components.

Venom of the honeybee Apis mellifera is a toxic substance which is known to man for thousands of years. However, the venom composition as well as its mode of action started to be elucidated in the 1960s(2,29).

The components of bee venom can be separated according to its molecular weight, obtaining then different fractions composed of enzymes (hyaluronidase and phospholipase A2), mellitin, oligopeptides (secapine, mast cell degranulation peptide (MCD), tertiapine and apamine) procamine and low-molecular-weight compounds (histamine, dopamine, noradrenaline and others)(20).

Couch and Benton(17) reported on a great interest in the production of Apis mellifera venom in the USA. The venom would be used both for desensitization of hypersensitive individuals and for the treatment of rheumatoid arthritis. According to Hyre and Smith(33), the efficacy of bee venom for the treatment of rheumatoid arthritis has been extensively reported, although the precise mode of action is not yet known. Reports on the literature say that the venom induces production of corticosterone, release of histamine and exhibits antiinflammatory activity. Billingham et al.(11) isolated the MCD peptide of bee venom which is believed to be responsible for the antiinflamatory activity.

In addition to its antiinflammatory action, the venom of Apis mellifera is a radioprotective agent. Shipman and Cole(59) verified that mice subcutaneously injected with bee venom 24 h prior to irradiation with a lethal dose of X-ray (850R) presented a survival rate of 64% in 30 days.

Kanno et al.(38) conducted other experiments using mice weighing approximately 30g. These animals were subcutaneously injected with 2.8 or 5.6µg/g of body weight of bee venom prior to whole body exposure to 937 R of gamma radiation of cobalt-60. The non-treated irradiated mice presented 100% of mortality 15 days after irradiation. Survival around the 60 day was of 10% for the group that received 2.8µg/g and of 18% for the group that received 5.6µg/g of body weight. The animals treated only with the venom showed discreet pathological alterations in the liver and bone marrow due to irradiation. In such organs, direct toxic effects caused by the venom were not observed. However, it is believed that the venom is toxic to the kidneys due to degeneration of renal epithelium, and it also seemed to cause hemolysis.

In 1975, Artemov et al.(3) made a preparation using bee venom without high-molecular weight proteins and enzymes which are usually present in the venom. This "venom" was called apilite. A high survival rate was obtained in groups of mice which were subcutaneously injected with a dose of 6µg/g of body weight of apilite prior to radiation exposure. The same protective effect was not observed after administration of the same dose of apilite right after irradiation or after administration of 1µg/g of body weight/day during 10 days following irradiation.

Some of the venom compounds were tested separately for their radioprotective action. Ginsberg et al.(27) conducted a series of experiments in which mellitin was subcutaneously injected in rats 24 h prior to irradiation (8.5 Gy). These authors verified that the animals survived up to 30 days after receiving a dose of 60 mg/kg of body weight of mellitin. However, a good protection was achieved with doses of approximately 5 mg/kg of body weight. This considerable discrepancy between the maximum tolerated dose and the effective dose indicate that radioprotection conferred by mellitin is pharmacologically induced.

Histamine is one of the venom components which was also tested for its possible radioprotective action. The two histamine-terminal peptides found in bee venom such as alanylglycylglutaminylglycylhistamine procamine and alanylglycylprolylalanylglutaminylhistamine might release histamine in vivo by being slowly hydrolyzed and models suggest that both might release chelate copper (Cu ) ions. Thus, these compounds might also contribute to the radioprotective properties of natural venom.(51)

To test the hypothesis that histamine-terminal peptides might exhibit radioprotective activity, Peck et al.(52) initiated studies with compounds named glycylhistamine, analogous to histamine-terminal found in the venom. They found that glycylhistamine afforded radioprotection when administered intraperitoneally 30 minutes before exposure in the same manner as a subcutaneous injection of 2.75 mg/kg of body weight of whole venom administered 24h prior to irradiation (60% of survival). However, the strongest protection was obtained when glycylhistamine dihydrochloride (1000 mg/kg) was injected subcutaneously 24h prior to irradiation with 75% of survival. Differences noted between the 24h and 30 minute i.p. injections might be due to excretion or metabolic alteration of glycylhistamine or its hydrolysis product, while the significant improvement in radioprotection associated with subcutaneous injection is consistent with the low absorption typical of this method of administration. The results obtained are encouraging and suggest further research about glycylhistamine and other analogs. The low toxicity and long delay properties of glycylhistamine administered subcutaneously indicated that this compound can probably afford protection for mammals against a semi-lethal dose of ionizing radiation.

According to Shipman and Cole(59), bee venom belongs to a class of radioprotectors known as biostimulants, since it produces alterations in the body's physiological state. For this reason, bee venom is effective only when injected one or more days before irradiation. These authors tested the radioprotective property of Apis mellifera venom administered subcutaneously (4.3 mg/kg of body weight and 50 mg/kg of body weight) in mice irradiated with a lethal dose of X-rays. They observed a significantly higher number of surviving animals when venom was administered 24h prior to irradiation. However, the same result was not observed when the venom was administered 30 minutes or right after exposure to irradiation. Therefore, bee venom differs from the classical radioprotectors such as cysteine, cysteamine or AET, which are effective only when administered about 30 minutes prior to exposure to radiation. Shipman and Cole(59) reported that Apis mellifera venom might produce stress in animals, and thereby, elicit the so-called "adaptation syndrome" which would increase radioresistance. Venom may also have antibacterial property and reduce radiation effects or even produce alterations in the hematopoietic system common to biostimulant radioprotectors such as interleukins.

According to Barrett and Stockham(8) the stress might cause an increase of radioresistance due to a rise in corticosterone levels within 15 minutes.

Couch and Benton(17) showed in rats that the highest levels of corticosterone were detected 1 h after bee venom injection. Varanda et al.(74) rejected this hypothesis because they did not detect radioprotection when the venom was administered 1 h prior to radiation.

The second hypothesis which suggests that bee venom has an antibacterial property was also discarded by Varanda et al.(74), since the period of 24 h (period when significant radioprotection was obtained) is not enough for the development and block of an infectious condition.

The third mechanism involving changes in the hematopoietic system was considered valid by Varanda et al.(74), since Hyre and Smith(33) carrying out in vitro and in vivo experiments observed that bee venom produced changes on functions of T and B lymphocytes in BALB/c mice. In addition, according to Asaoka et al.(4), the release of precursors of pro-inflammatory mediators caused by the administration of phospholipase A2 might increase the cellular response of T lymphocytes.

Ginsberg et al.(27) also noticed an increase in mouse survival when mellitin was administered subcutaneously 24 h prior to exposure to 8.5 Gy of X-rays. This radioprotective effect of mellitin differs from other radioprotetors in at least two aspects. First, most of the radioprotective compounds are relatively simple substances and not structural proteins; secondly, these substances are efficient only when administered approximately 30 minutes prior to irradiation.

Although Shipman and Cole(59) and Ginsberg et al.(27) have reported the radioprotective effects of bee venom and mellitin, they used the animals' survival time as a parameter which was measured using a very small sample of 15 animals. Varanda et al.(74) demonstrated the radioprotective property of Apis mellifera whole venom when administered intraperitoneally 24h 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 234 (46.8%) in the group that only received radiation to 68 (14.4%) in the group that was exposed to radiation and received venom, presenting a protection magnitude around 70%. In addition, 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 venom 24 h prior to radiation. Protection magnitude was of 57.5%.

The radioprotective effect of Apis mellifera venom was also noted by Varanda and Takahashi(73) in in vitro studies with human peripheral blood lymphocytes irradiated with 2.0 Gy of gamma radiation.

Costa and Takahashi(16), studying the radioprotective effect of mellitin and phospholipase A2 of Apis mellifera venom on bone marrow cells of Wistar rats observed a decrease in radiation-induced chromosomal damage, though this decrease was not statistically significant. The authors believe that mellitin and phospholipase A2 somehow contribute to radioprotection conferred by Apis mellifera whole venom.

Within this context, the venom of Apis mellifera is believed to have radioprotective activity due mainly to the stimulation of the hematopoietic system. However, other mechanisms such as release of histamine induced by the MCD peptide and reduction in blood oxygen tension induced by phospholipase A2 also are factors which contribute to the radioprotective effect of bee venom.

REFERENCES

01 AINSWORTH EJ. From endotoxins to newer immunomodulators: survival-promoting effects of microbial polysaccharide complexes in irradiated animals. Pharmacol. Ther., 1988, 39, 223-41.

02 ARTEMOV N.M. The bee venom as a produce of the apiculture. Apimondia. In: International Beekeep Jubilee Congress, 2, Bucharest, 1965.

03 ARTEMOV NM., KON'KOVA LG., SERGEEVA LF. The effect of Apilite on the radiosensitivity of mice. Radiobiology, 1975, 15, 462-5.

04 ASAOKA Y., YOSHIDA K., SASAKI Y., NISHIZUKA Y., MURAKAMI M., KUDO I., INOUE K. Possible role of mammalian secretory group II phospholipase-A2 in T-lymphocyte activation implication in propagation of inflammatory reaction. Proc. Natl. Acad. Sci. USA,1993, 90, 716-9.

05 BACQ ZM. Chemical protection against ionizing radiation. Spingfield: THOMAS CC., 1965. 8-12.

06 BACQ ZM., DENECHAMPS G., FISHER P., HERVE A., LEBIHAN H., LECOMTE J., PIROTTE M., RAYET P. Protection against X-rays and therapy of radiation sickness with b-mercaptoethylamine. Science, 1953, 117, 633-6.

07 BACQ ZM., HERVE, A. Ein chemischer Schutz gegen Röntgenstrahlungen. Strahlentherapie, 1954, 95, 215-37.

08 BARRETT AM., STOCKHAM MA. The effect of housing conditions and simple experimental procedures upon the corticosterone level in the plasma of rats. J. Endocrinol., 1963, 26, 97-106.

09 BETZ EH. Contribution a l'étude du syndrome endocrinien provoqué par l" irradiation totale de l" organisme. Liège: Thone, Liège, 1955.

10 BIAGLOW. JE. The effects of ionizing radiation on mammalian cells. In: FARHATAZIZ RODGERS MAJ. Ed. Radiation chemistry: principles and applications. New York: VCH, 1987: 527-63.

11 BILLINGHAM MEJ., MORLEY J., HANSON JM., SHIPOLINI RA., VERNON CA. An anti-inflamatory peptide form bee venom. Nature,1973, 245, 163-4.

12 BROWN PE. Mechanism of action of aminothiol radioprotectors, Nature, 1967, 213, 363-4.

13 CHIRIGOS MA., PATCHEN ML. Survey of newer biological response modifiers for possible use in radioprotection. Pharmacol. Ther.,1988, 39, 243-6.

14 CHO ES., JOHNSON J., SNIDER BCF. Tissue glutathione as a cystine reservoir during cystine depletion in growing rats. J. Nutr., 1984, 114, 1853-62.

15 CHORVATOVICOVA D. Radioprotective effect of stobadine in the mouse micronucleus test. Mutat. Res., 1994, 324, 7-11.

16 COSTA CTA., TAKAHASHI CS. Efeito modulador dos componentes do veneno de abelha sobre os danos citogenéticos induzidos pela radiação gama em ratos Wistar. Ribeirão Preto: USP, Faculdade de Medicina, Departamento de Genética e Matemática Aplicada à Biologia, 1995. 70p. [Dissertação - Mestrado]

17 COUCH TL., BENTON AW. The effect of the venom of the honey bee, Apis mellifera L. on the adrenocortical response of the adult male rate. Toxicon,1972, 10, 55-62.

18 DALE WM. The effect of X-rays on the conjugated protein d-amino-acid oxidase. Biochem. J.,1942, 36, 80-5.

19 DELANEY JP., BONSACK ME., FELEMOVICIUS I. Lumeral route for intestinal radioprotection. Am. J. Surg.,1993, 166, 492-501.

20 DOTIMAS EM., HIDER, RC. Honeybee venom. Bee world,1987, 68, 51-70.

21 EASTGATE J., MOREB J., NICK HS., SUZIKI K., TANIGUCHI N., ZUCALI JR. A role for manganese superoxide dismutase in radioprotection of hematopoietic stem cells by interleukin-1. Blood.,1993, 81, 639-46.

22 EL-NAHAS SM., MATTAR FE., MOHAMED AA. Radioprotective effect of vitamins C and E. Mutat. Res.,1993, 301, 143-7.

23 FEDOROCKO P., BREZÁNI P., MACKOVÁ NO. Radioprotection of mice by the bacterial extract Broncho-Vaxom: haemopotetic stem cells and survival enhacement. Int. J. Radiat. Biol., 1992, 61, 511-8.

24 FEDOROCKO P., BREZÁNI P., MACKOVÁ NO. Radioprotective effects of WR-2721, Broncho-Vaxom, and their combinations: survival, myelopoietic restoration, and induction of colony-stimulating activity in mice. Int. J. Immunopharmacol., 1994, 16, 177-84.

25 FELEMOVICIUS I., BONSACK ME., BATISTA ML., DELANEY JP. Intestinal radioprotection by vitamin E (alpha-tocopherol). Ann. Surg., 1995, 222, 504-10.

26 FLOERSHEIM GL. Radioprotective effects of calcium antagonists used alone or with others types of radioprotectors. Radiat. Res.,1993, 133, 80-7.

27 GINSBERG NJ., DAUER M., SLOTTA KH. Melitin used as protective agent against X-irradiation. Nature,1968, 220, 1334.

28 GUPTA R., UMA DEVI P. Protection of mouse chromossome against whole body gamma irradiation by sulphydryl compounds. Br. J. Radiol., 1986, 59, 625-7.

29 HABERMANN E. Bee and wasp venoms. Science,1972, 177, 314-22.

30 HELD KD., AWAD S. Effects of polyamines and thiols on the radiation sensitivity of bacterial transforming DNA. Int. J. Radiat. Biol.,1991, 59, 699-710.

31 HELTON WS. The pathophysiologic significance of alterations in intestinal permeability induced by total parenteral nutrition and glutamine. J. Parenter. Enteral Nutr., 1994, 18, 289-90.

32 HORIUCHI K., MIYAMOTO T. Radioreductive effect of bestatin (ubenimex) in BALB/c mice. Int. J. Radiat. Biol.,1992, 62, 72-80.

33 HYRE HM., SMITH RA. Immunological effects of honey bee (Apis mellifera) venom using Balb/c mice.Toxicon, 1986, 24, 435-40.

34 JOHNSTONE PAS., DeGRAFF WG., MITCHELL JB. Protection from radiation-induced chromosomal aberrations by the nitroxide tempol. Cancer,1995, 75, 2323-7.

35 KALECHMAN Y., ALBECK M., ORON M., SOBELMAN D., GURWITH M., SEHGAL SN., SREDNI B. The radioprotective effects of the immunomodulator AS101.J. Immunol., 1990, 145, 1512-7.

36 KALECHMAN Y., GAFTER U., SOTNIK-BARKAI I., ALBECK M., GURWITH M., HORWITH G., KIRSCH T., MAIDA B., SEHGAL SN., SREDNI B. Mechanism of radioprotection conferred by immunomodulator AS101. Exp. Hematol., 1993, 21, 150-5.

37 KALECHMAN Y., ZULOFF A., ALBECK M., STASSMANN G., SREDNI B. Role of endogenous cytokines secretion in radioprotection confered by the immunomodulator Ammonium trichloro (dioxy ethylene-0-0") tellurate. Blood, 1995, 85, 1555-61.

38 KANNO I., ITO Y., OKUYAMA S. Radioprotection by bee venom. Nippon Igaku Hoshasen Gakkai Zasshi, 1970, 29, 1494-500.

39 KLIMBERG VS., SALLOUM RM., KASPER M., PLUMLEY DA., DOLSON DJ., HAUTAMAKI RD., MENDENHALL WR. Oral glutamine accelerates healing of the small intestine and improves outcome after whole abdominal radiation. Arch. Srg.,1990, 125, 1040-5.

40 KLIMBERG VS., SOUBA WW., DOLSON DJ., SALLOUM RM., HAWTAMAKI RD., PLUMLEY DA., MENDENHALL WM. Prophylatic glutamine protects the intestinal mucosa from radiation injury. Cancer,1990, 66, 62-8.

41 KLIMBERG VS., SOUBA WW., SALLOUM RM. Glutamine-enriched diets support muscle glutamine without stimulating tumor growth. J. Surg. Res., 1990, 48, 319-23.

42 LANGENDORFF H., KOCH, R. Untersuchungen über einen biologischeb strahlenschutz IX. Zur Wirkung von SH - Blockern auf die Strahlenempfindlichkeit. Strahlentherapie, 1954, 95, 531-41.

43 LITTLEFIELD LG., HOFFMANN RG. Modulation of the clastogenic activity of ionizing radiation and bleomycin by the aminothiol WR-1065. Environ. Mol. Mutagen.,1993, 22, 225-30.

44 MACKOVÁ NO., FEDOROCKO P. Combined radioprotective effect of Broncho-Vaxon and WR-2721 on hemopoiesis and circulating blood cells. Neoplasma, 1995, 42, 25-30.

45 MEFFERD RB. JR, HERKEL DT., LOEFER JB. Effect of Piromon on survival in irradiated mice. Proc. Soc. Exp. Biol. Med.,1953, 83, 54-6.

46 MILAS L., HUNTER N., STEPHENS LC., PETERS LJ. Inhibition of carcinogenesis in mice by S-2-(3-aminopropilamina)-ethyl phosphorothioic acid. Cancer Res.,1984, 44, 5567-9.

47 NETA R., OPPENHEIM JJ., WANG JM., SNAPPER CM., MOORMAN MA., DUBOIS CM. Synergy of IL-1 and stem cell factor in radioprotection of mice is associated with IL-1 up-regulation of mRNA and protein expression for c-kit on bone marrow cells.J. Immunol., 1994, 153,1536-43.

48 NOWOTNY A., BEHLING UH., CHANG HL. Relation of structure to function in bacterial endodotoxins VIII. Biological activities in a polysaccharide rich fraction. J. Immunol.,1975, 115, 199-203.

49 PATT HM. Protective mechanisms in ionizing radiation injury. Physiol. Rev.,1953, 33, 35-76.

50 PATT HM., TYREE EB., STRAUBE RL., SMITH DE. Cysteine protection against X-irradiation. Science, 1949, 110, 213-4.

51 PECK ML., O'CONNOR R. Procamine and other basic peptides in the venom of the honeybee (Apis mellifera). J. Agric. Fd. Chem., 1974, 22, 51-3.

52 PECK ML., O'CONNOR R., JOHNSON TJ., ISBELL AF., MARTELL AE., McLENDEN G., MEFF RD., WRIGHT DA. Radioprotective potential and chelating properties of glycylhistamine an analog of histamine terminal peptides found in bee venom. Toxicon, 1978, 16, 690-4.

53 PHIL A., ELDJARN L. Pharmacological aspects of ionizing radiation and chemical protection in mammals. Pharmacol. Rev.,1958, 10, 437-74.

54 PODLESKI WK. Immunomodulation of allergic autocytotoxicity in bronchial asthma by a bacterial lysate-Broncho-Vaxon. Int. J. Immunopharmacol., 1985, 7, 713-8.

55 PRAGER A., TERRY NH., MURRAY D. Influence of intracellular thiol and polyamine levels on radioprotection by aminothiols. Int. J. Radiat. Biol., 1993, 64, 71-81.

56 PRASAD KN. Acute radiation syndromes. In PIZZARELO, DL. Ed. Radiation biology, Boca Raton: CRC, 1982: 205-35.

57 REAL A., GÜENACHEA G., BUEREN JA., MAGANTO, G. Radioprotection mediated by the haemopoietic stimulation conferred by AM5: a protein associated polysaccharide.Int. J. Radiat. Biol., 1992, 62, 65-72.

58 SARMA L., KESAVAN, PC. Protective effects of vitamins C and E against gamma-ray induced chromosomal damage in mouse. Int. J. Radiat. Biol.,1993, 63, 759-64.

59 SHIPMAN WH., COLE LJ. Increased radiation resistence of mice injected with bee venom one day prior to exposure. Nature, 1967, 215, 311-2.

60 SMITH LH., CONGDON CC. Experimental treatment of acute whole-body radiation injury in mammals. In. HOLLAENDER A. Ed. Radiation protection and recovery. New York: Pergamon , 1960, 242-302.

61 SMITH LH., MACKINLEY JR. TW. Mechanisms of radioprotection of mice by phenyldrazine. Radiat. Res.,1972, 50, 611-28.

62 SMITH WW., ALDERMAN IM., GILLESPIE RE. Increased survival of irradiated animals treated with bacterial endotoxins. Am. J. Physiol.,1957, 191, 124-30.

63 SMITH WW., BRECHER G., RUDD RA., FRED S. Effect of bacterial endotoxin on the occurrence of spleen colonies in irradiated mice. Radiat. Res., 1966, 27, 369-74.

64 SMITH WW., BUDD RA., CORNFIELD J. Estimation of radiation dose-reduction factor for ß-mercaptoethylamine by endogenous spleen colony counts. Radiat. Res., 1966, 27, 363-8.

65 SNYDER SL., WALDEN TL., PATCHEN ML., McYITTIE TJ., FUCHS P. Radioprotective properties of detoxified lipid A from Salmonella minnesota R 595. Radiat. Res.,1986, 107, 107-14.

66 SOUBA WW., KLIMBERG VS., COPELLAND EM. Glutamine nutrition in the management of radiation enteritis.J. Parenter. Enteral Nutr., 1990, 14, 1065-85.

67 SPOTHEIM-MAURIZOT M., RUIZ S., SABATTIER R., CHARLIER M. Radioprotection of DNA by polyamines. Int. J. Radiat. Biol.,1995, 68, 571-7.

68 SREDNI B., CASPI RR., KLEIN A., KALECHMAN Y., DANZINGER Y., BEN YAAKOV M., TAMARI T., SHALIT F., ALBECK M. A new immunomodulating compound (AS-101) with potential therapeutic application.Nature, 1987, 330, 173-6.

69 TABOR CW., TABOR H. 1,4-Disminobutane (putrescine), spermidine, and spermine. Ann. Rev. Biochem.,1976, 45, 285-306.

70 TAVARES DC., TAKAHASHI CS. Effects of the amino acid glutamine on frequency of chromosomal aberrations induced by gamma radiation in Wistar rats. Mutat. Res., 1996, 370, 121-6.

71 URBASCHEK R. Effects of bacterial products on granulopoiesis. Adv. Exp.Med. Biol., 1980, 121, 51-64.

72 VAN BEKKUM DW., DeVRIES MJ. Radiation Chimaeras. London: Logos/Academic Press, 1967.

73 VARANDA EA., TAKAHASHI CS. Effect of pretreatment with venom of Apis mellifera bees on the yield of gamma-ray induced chromosome aberrations in human blood lymphocytes. Rev. Bras. Genet., 1993, 16, 551-9.

74 VARANDA EA., TAKAHASHI CS., SOARES AEE., BARRETO SAJ. Effect of Apis mellifera bee venom and gamma radiation on bone marrow cells of Wistar rats treated in vivo. Rev. Bras. Genet., 1992, 15, 807-19.

75 WINTERS R., MATTHEWS R., ERCAL N., KRISHNAN K. Glutamine protects chinese hamster ovary cells from radiation killing. Life Sci., 1994, 55, 713-20.

76 ZUCALI JR., MOREB J., GIBBONS W., ALDERMAN J., SURESH A., ZHANG Y., SHELBY B. Radioprotection of hematopoietic stem cells by interleukin-1. Exp. Hematol., 1994, 22, 130-5.

Received 16 October 1996

Accepted 16 November 1996

CORRESPONDENCE TO:

E. A. VARANDA - Departamento de Ciências Biológicas, Faculdade de Ciências Farmacêuticas de Araraquara, Rodovia Araraquara-Jaú, Km 01, CEP 14.801-902, Araraquara, São Paulo, Brasil.

Publication Dates

• Publication in this collection
  26 Nov 1998
• Date of issue
  1998

History

• Accepted
  16 Nov 1996
• Received
  16 Oct 1996