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Abstract Fractionation of N. nigricoJJis, N. nivea, N. meJanoJeuca and N. haJe venoms using ion-exchange chromatography on Amberlite CG-50 and ammonium bicarbonate elution gradient, yielded, 13, 12, 11 & 7 different fractions respectively. There were 2, 4, 4 & 2 lethal fractions for N. nigricoJJis, N. nivea, N. haJe venoms. N. nigricoJJis venom caused heamorrhagic patched at the site N. meJanoJeuca & of inj ection, lungs and stomach; this activity was only shown with some of the other venoms fractions when injected doses. in higher The i.m LD”,., in mice were 0.235::,0.042, 0.45::,0.073, 1.75::,0.154 & 3.0::,0.269 mg/kg for N. melanoleucd, N. n i v e e , N. haJe and N. nigricoJJis venoms respectively. The dose mortality curves for the 4 venoms were nearly parellel, revealing a possible similarity in the causes of death. The 4 NaJa venoms studied showed similar effects on the isolated guinea-pig heart and atrium. The venoms caused marked negative inotropic and chronotropic effects followed by contracture which terminated in complete cardiac arrest. The effects were not reversed by extensive washing and not blocked by either hexamethonium, or atropine excluding the possibil ity of a cholinergic component in their action. Calcium chloride, however, completely antagonised the venom-induced contracture and cardiac depression. It was concluded that binding of the cardiotoxins in the venoms is inhibited by increased Ca++. The most common electrocardiographic effects of the venoms were, bradycardia, AV-block, ischemia and inferior wall in farction. The bradycardia is probably due to the direct depressant effect of the venoms as was evidenced from the results on the isolated preparations • Various types of AV-block were produced. .First degree heart block in the early phases and second degree heart block in the later phases of envenomation. This indicated interference by the venoms of the conducting system at different levels in the AV-bundle. Myocardial ischemia was evidenced from depression of ST-segment particularly in I & aVL. This was supported by the decrease in perfusion rate of coronary flow in the isolated hearts. Inferior wall infarction was evidenced from the elevation of ST segment in II, III & aVF in all venoms except N. nivea where elevation the ST segment occured in III & Vl. In addition, the T wave was inverted in different leads including I , II, aVL, Vl & V5. With the exception of N. nivea venom, the three other venoms induced a tall peaked and slender T wave in all leads indicating the presence of hyperkalemia. It is worth mentioning here that N. nivea venom was the least potent in causing electrocardiographic changes and in decreasing coronary flow. On a weight bases, N. melanoleuca venom was the most potent in causing the electrocardiographic effects. Incubating venoms with their specific antivenoms completely prevented venoms-induced cardiac fects. and electrocardiographic ef- In the present study, some of the NaJa venoms caused a rise or fall in the arterial blood pressure of the anaesthetized cat. This may be due to release of medullary tamine and or acetylcholine. catecholamines, I his- The action of Naja venoms on the nerve muscle pr parations was mainly confined to the cholinergic neuroeffector tr nsmission sites. The venoms bind strongly and specifically to the cholinergic motor end plate and the blocking effect was not reversed by washing. The Naja venoms block the transmission mainly by a postjunctional mechanism. This was evidenced from the protection from the blocking effect of the venoms by the competitive neuromuscular blocking drugs, d-tubocurarine or gallamine. The protection was related to the bulk of the drug, its better fit to the receptors and the number of onium groups as better protection was achieved with gallamine than with d-tubocurarine. The complete protection from the block by succinylcholine was unexpected. This effect is probably due to binding of succinylcholine to the receptors and because .of its aliphatic chain residue, the drug is expected to possess a very flexible molecule that can fit in the cholinergic receptor better than the rather rigid mplecules of dtubocurarine or gallamine. The block caused by the venoms was mainly of the non-depolarizing type as was shown from the absence of any contraction of the skeletal muscle preparations used on addition of the venoms, the flaccid paralysis of the conscious chicks injected with the venoms and that the intensity of blockade was greater at higher frequencies of nerve stimulation or following application of tetanus. Neostigmine caused a transient reversal of the venom-induced blocking activity in the phrenic nerve-hemidiaphragm and partial recovery of the response to acetylcholine in rectus abdominis muscle. Tetraethlyammonium did not antagonise the blocking action of the venoms on the phrenic nerve-hemidiaphragm but restored a partial recovery after venoms in the rectus abdomins preparation. The effect of both drugs is probably caused by increasing the concentration of acetylcholine which in turn antagonizes the competitive neuromuscular blocking action of the venoms. These effects refer to a possible presynaptic mechanism in the ac tion of the venoms. neuromuscular Choline did not antagonise the venom-induced blockade in both the phrenic nerve-hemidiaphragm and rectus abdominis. This suggests that NaJa venoms either do not act on choline transport mechanism or that any action at this site is none-competitive in nature. Naja venoms act partly through depolarization as well maintained tetanus was obtained during transmission failure. In this respect N. nigricollis venom caused spastic paralysis in the conscious chick and contracture of the phrenic nerve-hemidiaphragm. Also, both N. nigricollis and N. haJe venoms caused contracture of the chick biventer cervicis muscle probably due to their contents of cardiotoxins. The contracture might also be due to the release of acetylcholine. Lowering the Ca++ concentration in the Krebs solution enchanced the blocking effect induced by the NaJa venoms on the phrenic nerve-hemidiaphragm. The effects of N. nigricollis and N. haJe venoms were particularly affected in this ~espect. On the contrary, lowering the magnisum ion concentration caused a par- tial protection from the venoms effect. The effect was particularly significant in case of N. nigricollis and N. nivea venoms. This indicates that venoms studied may interfer with the release mechanism. The most lethal fractions were those possessing neuromuscular blocking effect; their blocking effect was prevented by pretreatment of the muscle preparatons with d-tubocurarine or gallamine denoting their a-neurotoxin nature. They are FII and FV of N. haje, FII of N. nigricollis, FVI of N. melanoleuca venoms. Incubating the venoms with their spicific antivenoms, FIV and FVII of N. nivea and completely prevented their neuromuscular blocking effect. The 4 Naja venoms caused stimulation of the isolated guineapig ileum, an effect which was subject to tachyphylaxis. The action may be due to the release of either acetylcholine or histamine. In higher concentrations all venoms decreased the size of the twitches of the field stimulated guinea-pig ileum and the response to subsequent doses of acetylcholine. Pretreatment with morphine did not influence the inhibition. This indicates that the venoms in’the doses used are either more potent than morphine in inhibiting the release of acetylcholine, or they inhibit the release of the transmitter by a mechanism different of that of morphine. Only N. nigricollis venom antagonised the effect of histamine on the isolated guinea-pig ileum. The antagonism is likely due to the stimulation of the sympathatic nerve endings. Naja venoms stimulated rat uterus in both de Jalon and ringer locke solutions, an effect which was subject to tachyphylaxis. The stimulant effect was not blocked by hexamethonium, atropine, cyproheptadine or indomethacin excluding the possibility of being mediated via choline, serotonine or prostaglandins. the release of acetyl- The effect of N. nivea venom on the spontaneously contracting rat uterus was an exception since it was blocked by either cyproheptadine or indomethacin. Also the effect of N. melanoleuca venom on the uterus in de jalon solution was exception since it was blocked by indomethacin. Meclofenamic acid completetly antagonised the spasmogenic effects of all NaJa venoms studied which suggests that NaJa venoms act through release of kinins or slow reacting substanc (SRSA). All NaJa venoms attenuated the response of rat uterus to serotonin. This may be attributed to an a-adrenergic blocking effect. NaJa venoms reduced the size of the twitches of the field stimulated rat vas deference. They also attenuated the response to the exogenously added norepinephrine. Pretreatment with indomethacin, antagonised the inhibitory effect of N. melanoleuca venom. This indicates that the action of the venom be due to release of prostaglandines. In the reserpine treated rat vas deferens, the inhibitory effect of NaJa venoms was pronounced and led to complete blockade of twitch activity. This effect is attributed to the a-adrenergic blocking effect of the venoms. Plotting of blood radioactivity of the labelled NaJa venoms or their labelled toxins mixed with non-labelled venom vs time revealed a triexponential behaviour characteristic of a three compartment open-model. The values for the distribution halflives (t~ n) associated with the rapidly equlibrating compartment ranged from 3 to 5 minutes. These values reflect the very rapid uptake of the venoms and their toxins by the shallow compartment. On the other hand, the distribution half lives (t~ a) for the slowly equlibrating compartment, ranged from 22 to 47 minutes, thus indicating a much slower uptake. The overall elimination half lives (t~ S) ranged between 15 and 29 hours. N. had the shortest elimination half life (15 hours) among haJe toxin all the venoms and toxins studied. The graphical estimation of the equilibrium distribution ratios of the venoms and toxins concentrations in the shallow compartment to the central compartment (blood) are 0.5, 0.9; 0.8, 0.9; 1.6,1.0 and 1.7,1.0 for N. melanolecua, N. nigricollis, N. nivea and N. haJe venoms and their toxins respectively. This reveals a high affinity to the shallow compartment in case of N. nivea and N. haJe venoms and approximatly equal distribution of toxins in the shallow and central compartments. The ratios of venoms and toxins concentration in the deep compartment to the central compartment (blood) are 3.7, 4.0; 3.3, 3.4; 3.0, 1.7 and 1.3,2.7 for N. nigricollis, N. melanoleuca, N. nivea and N. haJe venoms and their toxins respectively. These values reflect the much higher affinity of the venoms and toxins to the deep compartment than to either the central or the shallow compartments. N. haje venom and N. nivea toxin showed the least affinity to the deep compartment compared to all venoms and toxins studied. The peak tissue levels in the shallow compartment was reached within 20-25 minutes following injection. However, most effects started 30-60 minutes. This indicates that the possible site of action of venoms or toxins are probably not located in the shallow compartment and would refer to the deep tissue compartment as the probable site of effect, as the peak tissue level in the deep compartment was reached 100-200 minutes following venom or toxin injection. The highest radioactivity was found in the thyroid gland followed by kidney, liver, lung and heart. Also very high radioactivity was found in the urine, bile and stomach contents. The very high activity found in the stomach contents would refer to the stomach as a possible route for elimination of the venoms. Also the high radioactivity found in the urine would refer to the kidney as the principal route for excretion and the bile as the next major route for elimination. All venoms and toxins showed a very high radioactivity in the thyroid gland except N. haje venom and its toxin which showed much lower values. This may be due to inhibition of the concentration mechanism for iodide by the thyroid tissue or to the possibility of their low metabolism. All venoms caused significant decrease in the gastric volume and free and total acidity together with a significant increase in the peptic activity. The ulceration and hemorrhagic patches observed follOWing venom injection are probably due to the increased peptic activity in all venoms and decrease of mucin. the significant Hyperimmunizing rabbits against N. nigricollis, N. mel.anoleuca, N. nivea and N. haje venoms or a mixture of the 4 venoms over a period of 4-8 months, yielded specific and polyvalent antivenoms of high potency. Using the immunodiffusion experiments, precipitin bands were obtained with venoms or venom fractions in concentrations as low as 10 mg/ml and using serum dilutions up to 1: 8. Also each antivenom showed definite and the precipitin bands with its own venom and its fractions, other venoms and their fractions. This indicates the presence of common antigens in the venoms. The antivenoms and the immunoglobulins separated were highly effective in neutralizing all the pharmacological effects induced by venoms. They also protected the mice against lethal doses equivalent to 5-50 times the LD~(:I. Except N. nivea vemom, the other three venoms casued hyperhypourecemia, hyper-uremia, hypophosphatimia hypocalcemia. The hyperglycemia could not be due and to The glycogenolysis since the rabbits were fasted for 17 hours. gluconeogenesis appeared to come mainly from lipolysis since urea level was not altered at times where blood glucose level was elevated. N. nivea venom on the other hand caused hypoglycemia, hyperurecemia, hypocalcemia and hypophosphatemia; serum urea and creatinine were not altered. This indicated that gluconeogenesis occurs mainly from lipolysis and not from amino acids. This would render the glucose generation lesser than in the case of the other three venoms. Also no muscular damage Dccured wiht N. nivea venom reflecting normal glucose utilization which coupled with decreased generation would lead to hypoglycemia. The decrease in serum uric acid caused by the 3 venoms is likely due to inhibition of uric acid synthesis. On the other hand the increased uric acid in case of N. nivea may be due to some components in the venom which may interfer with uric acid secretion in the renal tubules. The effect of the 4 NaJa venoms on serum sodium, potassium, calcium and phosphorous were nearly similar and reflects a picture of inhibion of the adrenal and parathyroid glands. The effects of the 4 NaJa venoms on total proteins, albumin and globulins are more or less similar and consisted of significant decrease possibly secondray to the haemorrhages induced in the lungs, stomach and intestine. Albumin loss is likely to be of renal origin. The different NaJa venoms caused a gradual increase in serum urea which was apparent only 12-24 hours. This increase was not associated with any significant increase. in serum creatinine or uric acid except with N. nive~ venom which caused an increase in uric acid. This indicates that the effect of NaJa venoms in the doses used on the kidney function is minimal. The NaJa venoms, with the exception of that of N. nivea, caused a significant increase in LDH, AST, ALT and CK activities reflecting a picture that correlates very well with the inferior wall infarction revealed in the electrocardiographic studies. The effect on CK activity reflects the action of the venoms on both myocardial and skeletal muscles. On the contrary, N. nivea venom decreased serum LDH, AST, ALT and CK activity, although the venom induced inferior wall infarction was evidenced from the results of the electrocardiographic studies. It is possible that N. nivea venom inhibited the activity of enzymes after their release by some components or released factors. All OnIy N. venoms seemed to increase ALT and decrease ALK.Phos. nigricollis venoms caused significant increased in bilirubin level which is likely due to the hemolytic effect of the venom. The effect of the venoms on the liver function appeared to be non-significant. All venoms increased serum chloride level. This may be due to metabolic acidosis and decreased chloride shift. An indirect evidence comes from the decreased volume and acidity of the gastric juice following venom injection. |