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INHIBITION OF PROTEIN SYNTHESIS BY AN EXTRACTOF THE VENOM SAC OF THE ORIENTAL HORNET

(YESPA ORIEIVTALIS)

rI'o whom reprint requeau should be addressed.

JOSEPH Fi:IEDMAN' and JACOB S. ISHAY~"'Department of Human (3enedcs and'Department of Physiolo8y and Pharmacolo8y, Sattler Faculty of

Medidne, Tel Aviv University, Raurat Aviv, Tel Aviv 69978, Israel

(Accrptedfor ptrblfcation 25 Novanber 1986)

J . Fatanrwv and J . S . %twx. Inhibition of protdn synthesisby as extras of the venom sac of theOriental horns (Yespa oriaetalis). Toxiooa 25, 673-676, 1987.-Venom from young and adulthornets inhibit protein ryathesi: in human lymphocytes and platelets, but does not affat thesynthesis of DNA and RNA. The venom activity is decreased by dialysis, and diminished more byboilin8 .

THE ORIENTAL hornet (Vespa orientalis), a social insect forming annual families, iswidespread in the Mediterranean basin and South East Asia (Bodenheimer, 1933). Thecontents and pharmacological activities of Oriental hornet venom sac extract have beeninvestigated by several authors (EDERY et al., 1972 ; ISHAY et al., 1974; SANDBANK et al.,1973 ; SLOB et al ., 1976 ; RING et al ., 1981). Thevenom contains relatively high molecularweight proteins such as hyaluronidaae, phospholipase and trehalase (EDERY et al., 1972).In addition, the venom sac extract contains dialysable lower molecular weight substancessuch as histamine, S-hydroxytryptamine, dopamine, adrenaline, noradrenaline, acetYlcholineand kinins (EDERY et al., 1972) . The lethality of the venom sac extract is related toontogenesis; the venom of young hornets (0-24 hr of age) is less toxic than the venomofadult (24 hr or older) hornets (ISHAY et al., 1977). In this study we used humanlymphocytes and platelets as convenient tools to follow the effect of hornet venom onDNA, RNA and protein synthesis.Hornets were collected by anesthetizing the entire colony at night, when all workers

were inside the nest . Thehornets were refrigerated to keep them immobilized and therebyenhance ease of handling and dissection . When time did not allow immediau dissection,the hornets were stored frozen at -20°C. The venom sac extract wasremoved by pullingcarefully on the sting. Saline was added to the collected sacs which were ground with amortar and pestle, centrifuged at 200 ~ for 10 min at 4°C and the supernatant stored at-20°C.Lymphocytes and platelets were obtained from the buffy coats of freshly drawnvenous

blood samples (anticoagulant : acid citrate/dextrose) from healthy donors . Mononuclear

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cells were separated by a Ficoll -hypaque gradient (BonrM, 1 %8). The collected cellswere washed with phosphate buffered saline and the platelets were separated fromlymphocytes by initial differential centrifugation at 150 g for 10 min and a furthercentrifugation at 480g for 10 min.To measure protein synthesis the lymphocytes and platelets were suspended in

phosphate buffered saline plus 1% glucose in a concentration of 10'/ml and 10°/Tnl,respectively . These 1 ml cell suspensions were preincubated with the venom for 30 min at37°C, then incubated for 2 hr in the presence of 1 1,~Ci r.-(4,S'H) leucine (45 Ci/mmole,Amersham, U.K.) . The cells were washed in phosphate buffered saline, centrifuged, andmacromolecules precipitated in 2 ml of 10% TCA. The precipitate was kept overnight at4°C, heated to 90°C for 15 min and centrifuged. The pellets were suspended in 0.2 ml of0.1 N NaOH andthen S ml of HydroLuma (Lumac, TheNetherlands) was addedandthesamples were counted in a Tricarb liquid scintillation spectrometer (Packard, Model3255).To measure RNA synthesis in lymphocytes the lymphocytes were suspended at a

concentration of 10'/ml, in Roswell Park Memorial Institute medium supplemented with15% heat inactivated fetal calf serum, antibiotics and glutamine (Biological Industries,Beth Haemek, Israel). Cell suspension of 1 ml were preincubated with the venom at 37°Cfor 30 min, then incubated for 2 hr in the presence of 1 pCi uridine-S'H (17 Ci/mmole,Negev Nuclear Research Center, Negev, Israel). The samples were washed in phosphatebuffered saline, precipitated in Solo TCA and filtered on fiberglass filters (GF/C, Tamar,Jerusalem, Israel), washed with 10% TCA. The filters were dried and placed in vialscontaining toluene scintillation mixture. The samples were counted as described above.To determine DNA synthesis in lymphocytes the lymphocytes were suspended at a

concentration of 106/ml in Dulbecco Modified Eagle medium supplemented with 15%heat inactivated fetal calf serum, antibiotics and glutamine. Two milliliters of the cellsuspensions were preincubated with the venom for 1 hr at 37°C, then incubated for onefurther hr in the presence of 10 ~Ci methyl'H -thymidine per ml (49 Ci/mmole, NegevNuclear Research Center, Negev, Israel). The cells were washed in phosphate bufferedsaline, precipitated in IONo TCA and treated the same as the RNA samples.The venom sac extracts of young hornets inhibited both the protein synthesis of

platelets (Table 1) and, along with the venom of adult hornets, the protein synthesis oflymphocytes (Table 2) . On the other hand, there was no inhibition of RNA or DNA

TAaLE 1. EFFECT' OF VENOM SAC EXTRACT FROM YOUNOHORNETS ON PROTEIY SYNT'I~SIS IN PLATELET'S

Venom

Results are shown as ~ of control in twoexperiments.

Control oounts/min values in Exps 1 and 2 were 4921and 1084, respectively .

(~ protein/ml) Exp. 1 Exp. 20 100 1008 15 118 + 1mM PMSF 13

20 7 8~ + 1mMPMSF 940 480 2

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TAaLE 2 . EFFECTS OF VENOM SAC EX7RACTS FROM YOUNG AND ADULT HORNETS ONPROTELN SYNIë~SIS IN LYMPHOCYTES

Venom

Results are shown as !~ of control in four experiments .Control counta/min values in Exps 1, 2, 3 snd 4 were 23,766, 83,964, 38,438 and

9322, respxtively .The results of three additional experiments are shown in Table 3 .

synthesis of lymphocytes in the presence of venom sac extracts in the range of 8-SO Ftgprotein/ml (data not shown) . Boiling markedly diminished venom activity, which wasonly partially diminished by dialysis (Table 3) .The addition of a protease inhibitor - phenylmethyl-sulfonyl fluoride (PMSF 1 mM)

- to the platelets or lymphocytes during their incubation with the venom of younghornets did not prevent the inhibition of protein synthesis (see Tables 1, 2) . Since in thevenom of young hornets the activities of phospholipase and protease are very low(ROSENBERa et al., 197, the present PMSF results suggest that proteolysis was not thecause of protein synthesis inhibition . Proteolysis, however, cannot be entirely ruled outsince other protease inhibitors have yet to be tested . Although the substances in the venomsac extract responsible for the observed effects are as yet unidentified, they are probablyproteins since their activity is inhibited by boiling. Some of these possibly acting ascofactors, are dialysable inasmuch as dialysis partially reduces the venom activity .

TAatB 3 . EFFECT ofsolLn~laANDDIALYSIS ofvErIOM sACBcTawcrFROMYOUNG HORNSß ON PROT8Q~19YN4i~.WS IN LYI~HOCY7~3

Results are shown as ~Fs of control in thra experl~ats.Control oounts/min values in Exps 1, 2 and 3 were 39,436, 65,741

sad 28,538, respectively .

Venom(Ng 1~rotdn/ml) ExP. i Exp . 2 ExP . 3Control 0 100 100 100Untr~ted 6 44 31 32

14 31 22 2328 25 19 14

Boiled 6 98 87 9614 75 65 8628 67 52 83

Dialysed 6 62 50 7314 34 10 3028 41 27 32

(~ protein/ml) Exp . 1 Exp . 2 Exp. 3 Exp . 4Control 0 100 100 100'Young' 8 4.6 37 30'Young' 8+1mM PMSF 3.6'Young' 20 0.7 43 43'Young' 40 24 23`Adult' 12 57 SO'Adult' 30 13 31Control 100'Young' 3 .1 43'Young' 7 .7 30'Young' 13 .5 13

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In mice, the LDP of adult hornet venom sac extract is approximately 70 mg/kg, whereasthat of young hornets is only about 2000 mg/kg. On the other hand, in our human cellsystem the younghornet venom maybe toxic because of its inhibition of protein synthesis .It may also be more toxic in animals upon repeated injections . The venom of the adulthornets serves, inter alia, as a predigesting agent, preparing the envenomated prey forconsumption. Venom of young hornets is not "capable" of predigesting, but, accordingto our data, it has the potential to inhibit protein synthesis, which apparently precedes thepredigesting potential of adult hornet venom. Under our experimental conditions, theinhibition of protein synthesis was unaccompanied by inhibition of DNA or RNAsynthesis, but one could expect DNA and RNA synthesis to be indirectly affected byprolonged protein synthesis inhibition. Such damage would depend on the rate ofdegradation of the various venom components and the half-lives of the proteins necessaryfor DNA or RNA synthesis. The effects of the extract of the venom sac resemble those ofspecific inhibitors of protein synthesis such as puromycin or cyclohexamide . There is apossibility that the effect of the venom sac extract is exerted at the translational level, i.e .assembly of ribosomes, initiation and/or elongation of peptide chains . We intend tofurther investigate hornet venom using a cell free system for protein synthesis, such aswheat germ (ROBERTS and PATERSON, 1983) or reticulocytes (PELHAM and JACKSON,1976).

ttr?t~tt~NCESBoDextma~e, F . S . (1933) Über die aktivitet von Vetpa orkntalis F . in Jahreaverlauf in PalBatina, ein Heitrag

fur i3kalogischea methodik. Zool. ayjBd. 102, S .HonnK, A . (1968) Isolation of mononuclear alla and granulocytes from human blood . Stand. 1. clin . lab.

Invrst . Suppl. 97, 21, 77 .EDHAY, H., ISHAY, J., Lwss, Y. and ßrrrea, S ., (1972) Pharmacological activity of Oriental hornet (Vetpa

orlattalit) venom. Toxirnn 10, 13 .IsftwY, J ., Lwss, Y ., B~-Sctucftwx, D., ßrrrea, S. and Swwnawrnc, U. (1974) The effects of hornet venom :ac

extract on the electrical activity of the cat brain. ToxJcon 12, 139.ISftAY, J ., Stiveo, A . sad ßrtrt=a, S . (1977) Lethality of venom sac extracts of the Oriental hornet (Vespa

orientalit) as related to ontogenesia. Twdcon 15, 307 .PEt.xwM, H. R . B. and Jwccsota, R. J . (1976) An efficient mRNA~ependmt translation system from

redculocyte lyaatea . Eur. J. Biochem . 67, 247 .Rio, B., Isxwv, J . and Stow, H . (1981) A neutral DNase in venom sac extract of the Oriental hornet Vespa

orie~etalis. Characterization, specificity and mode of action . Toxicon 19, 241 .RosEnTS, H . E. and PwreasoN, B . M . (1983) Efficient transladon of tobaao mosaic virus RNA and rabbit

globin 9S RNA in a cell-frasystem from commercial wheat germ . Prvc. natn. Aced. Scl. U.S.A . 70, 2330 .Roswaeaa, P., FsHwv, J . and ßrrren, S . (1977) Phospholipase A and B activities of the Oriental hornet (Vetpa

orientalis) venom and venom apparatus . Toxleon 1S, 141 .SwxnBwtvs:, U., Isttwx, J . and ßrrrea, S . (1973) Kidney changes in mice due to Oriental hornet (Yetpa'orientalis)venom : Histologic and elatron microscopy study . Acts pharrnac . tax. 32, 442.

Stow, H., Rnva, B . and IstiwY, J. (1976) Nucleases of the oriental hornet (Vespa orientalit) venom sac extract : I .Acid, neutral and alkaline deoxyrlbonucleases and their pharmacologial effects on cat blood in vitro.Toxicnn 14, 427 .