Teratogenicity in the rat of the venom from the scorpion Androctonus amoreuxi (Aud. & Sav.)

Toxrcon, Vol . 21, No . 2, pp . IT7 - 189. 1983 . Printed in Great Britain

0041 - 0101 /83/020177 - 12f03 .00/0 01983 Perpmon Pros Ltd.

TERATOGENICITY IN THE RAT OF THE VENOM FROM THE SCORPION ANDROCTONUS AMOREUXI (AUD . & SAV.) M. ISMAIL, A . C. ELLISON and A . K. TILMISANY Department of Pharmacology, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia (Amepted for publication 29 lune 1982) M . ISMAIL, A . C . ELLISON and A . K . TILMISANY . Teratogonicity in the rat of the venom from the scorpion Androttonus amoreuxi (Aud . 8c Sav .) . Taxiton 21, 177-189, 1983 . - A. amorcuxi venom caused a high foetal resorption rate in rats, particularly when injectod on days 9-11 of gestation . Vertebral and ossification defects and foetal weight loss were observed in many of the viable foetuses obtained from mothers treated with scorpion venom . Treatment of the rats with phentolamine in addition to the venom significantly roduced the venom-induced hyperglycemia . It also conferred some protection against foetal resorption but had only a slight effect on chondrification or foetal weight loss. This shows that hyperglycemia might be responsible for foetal mortality, but alone is not a decisive factor in the effect of the venom on the chondrification process . Treatment of the rats with triamterene reduced the foetal resorption rate and significantly decreased the effects of the venom on chondrification . However, marked stippling was observed in the long bones and was ascribed to marked mobilization of ionized calcium in the foetus . Foetuses removed from rats treated with phentohunine or triamterene in addition to the venom, however, showed flattened and depressed skulls, possibly from a missing 1st cervical vertebra or failure of the occiptal fontanel to close . Treatment of the rats with the scorpion venom over a logger period of time and starting at an earlier time of gestation (days 7 -14) caused total foetal resorption, which may be due to inhibition of histamine formation by the venom . The teratogenic effect of the venom appears to be the result of its metabolic effect and action on body electrolytes of the maternal animal, rather than to a direct effort on the foetuses. This was evidenced from experiments with labelled venom, where only a small fraction (0.08-0 .33%) was detected in foetuses or placenta .

INTRODUCTION IN GOATS, the repeated injection of small doses of venom from the scorpion Buthus minax caused incoordination of movements, progressive arching of the four limbs to the exterior and inability to stand without assistance . Autopsy revealed swelling of the heads of the long bones, especially in the knee joints (EL-ASMER et al., 1974, 1975) . Experiments in rats showed that B. minax venom decreased serum calcium and phosphorus and increased serum alkaline phosphatase. It decreased urinary excretion of calcium but increased that of phosphorus and inhibited intestinal absorption of calcium (EL-ASMAR et aJ., 1975 ; ISMAIL et al., 1978) . The effect of the venom on calcium metabolism, though marked, was not dramatic, possibly because fully grown animals were used in most cases . It was thought, therefore, that more pronounced effects might be obtained by using developing or young animals . B. minax and other scorpion venoms, however, are known to induce abortion in pregnant women and to stimulate the rat uterus, partly by direct action and partly through the release of kinins, prostaglandins and/or slow reacting substances) (OSMAN et al., 1972 ; ISMAIL et al., 1973, 1974a) . Venom from the scorpion Androctonus 177

17 8

M. ISMAIL, A. C. ELLISON and A. K. TILMISANY

amoreuxi is peculiar in causing relaxation of the rat uterus through stimulation of the ß-adrenergic receptors (GHAZAL et ai., 1975). A. amoreturi venom was chosen, therefore, as a model for studying the possible teratogenic effects of scorpion venom as related to calcium metabolism . MATERIALS AND METHODS Venom obtained from mature A. amoreuxi by electrical stimulation of the telson was suspended in water, centrifuged and the supernatant freeze-dried. Dried venom was kept in a desiccator over silica gel at -17°C and was reconstituted by the addition of saline or 0.5 M phosphate buffer, pH 7.5, immediately before use. Radioiodination of the venom was carried out as described by Isntwa. et al . (1980) . Virgin female (180-220 g) and male (220-250 g) Sprague Dawley rats bred in this laboratory were used . They were divided into groups of 4 females and 1 male, the males being allowed into the cages of the females from 6 p.m . to 9 a.m . each day. At 9 a.m . each day, vaginal smears were examined and rats showing a positive sperm test were judged pregnant . This was taken as day 0 of gestation. In the teratological studies, pregnant rats were divided into treatment groups of 12 -15 rats each . Group 1 was injected with scorpion venom 0.2 mg/kg i.p . daily for S successive days (days 8 - l2 of pregnancy) . Group 2 was injected with scorpion venom 0.2 mg/kg i.p . twice daily for a total of S doses (2 doses on each of days 9 and 10 and 1 dose on day 11 of pregnancy) . Group 3 was injected with scorpion venom 0.2 mg/kg i.p . daily for 8 successive days (days 7 -14 of pregnancy) . Groups 4 and 5 were treated with scorpion venom as forgroup 3, but group 4 was injected as well with phentolamine 1 mg/kg i.p . daily and group 5 was given triamterene 10 mg/rat orally daily, 30 min before injecting the scorpion venom. Group 6 was injected with saline and served as a control. All animals were observed daily for signs of gross toxicity. On day 20 of gestation the rats were anaesthetized with ether, blood samples withdrawn by cardiac puncture and foetuses removed by Caesarean section. The appearance of the placenta and the pelvic viscera of the mothers was noted. For each foetus, weight was rewrded and all gross abnormalities noted. Whole foetuses were fixed in absolute ethanol for 24 hr, eviscerated and then cleared in a 1 % aqueous solution of KOH for 24- 48 hr . The cleared foetuses were stained with 1 %a alizarin red in 1 % KOH solution for ?A hr and destained in a SO% solution of glycerine in water containing 1% KOH. The foetuses were then placed in glycerine and examined microscopically for ossification defects. Serum sodium, potassium and calcium in maternal blood were determined by atomic absorption spectrophotometry using a Varian dual channel double beam instrument type A-A-775. Maternal blood glucose was estimated using the glucose oxidase method Of SCHMIDT (1971) and test combination kits (Bcehringer, Mannheim, Germany) . Serum alkaline phosphatase in maternal blood was determined using the method of BessEY et al. (1946) and tat combination kits (Bcehringer, Mannheim, Germany) . Placental transfer of A. amoreuxi venom was also studied in rats on day 10, 11 and 20 of gestation. The rats were injected i.p . with "°I-labelled A. amoreuxi venom 1 mg/kg (specific activity 1 .2 NCi per mg). After 2 hr the rats were anaesthetized with ether, blood samples withdrawn by cardiac puncture and foetuses removed by Caesarean section. For the rats killed at day 20 of gestation, the amniotic fluid and placenta were also collected. A group of rats at day 11 of gestation was injected i.p . with 1 mg/kg of "'I solution in saline (specific activity 1 .2 ~Ci per mg) and served as a control. Radioactivity in foetuses, amniotic fluid and maternal blood, liver, kidney and thyroid was measured in biovials using a Beckman Gamma 4000 counting system with a 2 inch sodium iodide crystal (doped with thallium iodide) and connected to a Texas Instrument 700 printer. Statistical analysis of the results was performed using the Student t-test . Drugs: phentolamine (Regitine; Ciba, Switzerland) ; triamterene (Dyrenium; Smith Kline and French, Philadelphia, PA, U.S .A .) .

RESULTS

Table 1 shows that rats injected with scorpion venom daily for S successive days, starting at day 8 of gestation, exhibited a 58% foetal resorption rate . Injection of the same quantity of the venom, but giving the doses twice daily on days 9 and 10 and once on day 11 of gestation, caused 93% resorption of the foetuses . On the other hand, injecting the venom over a longer period and starting at an earlier time of gestation (days 7 -14) caused a 100% foetal resorption rate (Table 1). The average weight of the viable foetuses was significantly lower than that of the controls (P<0.05). Mortality rate in the treated rats ranged between 20 and 40% . When the rats injected with scorpion venom for 8 successive

Teratogenicity of Scorpion Venom T~a~ .E

1.

TERAT(XiENIC' EFFECT OF

Group

Treatment

Control (8) Venom-treated (8)

Saline

179

A. amoreux! vENCwt Total foetuses 63

Total Mean foetal viable wt in g1S.E .M foetuses 4.510 .32 61

0.2 mg/kg i.p . daily for 50 21 3 .04*0.21 " 5 successive days . (days 8-12). 45 3 3 .4910.11 " Venom-treated 0.2 mg/kg i.p . twice (5) daily (days 9 and 10) then once daily (day 11) . 43 0 Venom-treated 0.2 mg/kg i.p . daily for 8 successive days (days (5) 7-14). 49 9 3.4910.12" Phentolamine+ As with group (4) but venom-treated phentolamine 1 mg/kg (7) i.p . given 30 min before venom As with group (4) 47 6 2.6010.20" Triamterene+ but triamterene 10 venom-treated mg/rat orally given 30 min (8) before venom Figures in parentheses indicate number of rats in each group. Foetuses were removed by Caesarean section on day 20 of gestation. "Significantly different (P<0 .05) from the control group.

days were treated simultaneously with phentolamine, some protection was conferred to the foetuses, although the mortality rate in mothers was not markedly reduced. A foetal resorption rate of 81 .6% occurred in this group. Most animals developed soft stools and those who died had marked diarrhea before death. Treatment with triamterene simultaneously with scorpion venom also gave some protection to the foetuses, but did not reduce the mortality rate in the mothers. A foetal resorption rate of 87.2% occurred in this group. In both the phentolamine and the triamterene treated groups, the viable foetuses were significantly reduced in weight (Table 1). Most resorptions appeared to occur early in gestation, although some resorptions seemed to occur between days 10 and 20 of gestation. Foetuses removed from rats treated with phentolamine or triamterene in addition to scorpion venom showed flattened and depressed skulls . When the stained foetuses were examined, 27% of the foetuses removed from the rats injected with scorpion venom alone showed vertebral defects and 16% showed ossification defects involving the vertebral column and sternum (Table 2, Fig. 1). Ossification defects involving the fore and hind limbs were apparent in foetuses from rats treated with both venom and phentolamine, however no vertebral or rib defects were noted (Table 2, Fig. 2). Foetuses from rats treated with venom and triamterene showed spicule development within the long bones of the fore and hind limbs rather than well organized areas of ossification (Table 2, Fig. 2) . No rib defects were noticed in the triamterene-treated animals . The flattened skulls observed in both the phentolamine and triamterene treated groups may be the result of a missing 1 st cervical vertebra or faillue of the occipital fontanel to .close (Fig. 2). Analysis of the maternal blood samples withdrawn from the different treatment groups at day 20 of gestation showed that A . amoretixi venom had caused a significant lowering (P<0.05) of serum potassium and alkaline phosphatase (Table 3). Blood glucose was slightly lowered while serum sodium and calcium were not much affected (Table 3) .

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M. ISMAIL, A. C. ELLISON and A. K. TILMISANY

TABLE 2. OSSIFICATION DEFECTS IN VIABLE FOETUSES RECOVERED FROM MOTHERS TREATED WITH A. VENOM

Group Control (61)

Ne of foetuses showing normal or missing ossification centres in each group' No, of Fore limb Hind limb ossification Carpals Metacarpals Tarsals Metatarsals centres R L R L R L R L 0 1

0/ItOI~ClL1C1

Other defects R

L

2

Venom-treated 5 successive days (days 8 -12) (21) Venom-treated twice daily (days 9 & 10) and once daily (day 11) (3) Venom-treated 8 successive days (days 7 -14) Phentolamine+ venom-treated (days 7 -14) (9) Triamterene + venom-treated (days 7 - 14) (6)

3 4 0

1 2

3 4 0 1

100

100

10

16 66 18

90

100 100

100 100

100 12

88

100

100 100

17

87

100 100 Bent and defonmed ribs, shortened ribs, missing ribs, no tail . No defects noted, but only 3 out of 43 foetuses survived .

2

3 4 0 1

100

100

100

3 4 0 1 3 4 0 1

100

100

100

100

all resorbed

2

2

100

SO

50

25

73

13 37

50 50

12 25

63

SO 50

100

100

100 66

All foetuses had depressed or flattened skulls, possible absence of 1st 100 cervical vertebra . 100 Depressed or flattened skulls, bones lightly stained with scattered areas of ossification

SO 16 100 100 3 100 100 18 4 Venom was injected i.p . (0 .2 mg/kg). Phentolamine (1 mg/kg i.p .) was given 30 ntin . before venom. Triamterene (10 mg/rat orally) was given 30 min before venom. Foetuses were removed by Caeserean section on day 20 of gestation. Figures in parentheses indicate number of viable foetuses in each group. R,L,right and left, respectively . "Normal foetuses show 4 ossification centres in each of the carpals, metacarpals, tarsals and metatarsals. 2

Similar effects were obtained from rats treated with phentolamine or triamterene in addition to the venom. Rats treated with both triamterene and venom, however, showed a significant lowering of blood glucose (Table 3). An unusual, interesting finding observed in all pregnant rats treated with scorpion venom was a marked congestion of the pelvic blood vessels . The animals showed a large haematoma behind the rectum involving the area of the iliac, hypogastric and uterine arteries (Fig . 3) . Non-pregnant rats injected with scorpion venom and untreated pregnant rats did not show such findings (Fig. 3) . The distribution of the labelled venom in the foetuses and the maternal tissues at different times of gestation and of "°I at day 11 of gestation is shown in Table 4. Foetuses from rats injected with venom at days 10 and 11 of gestation showed significantly lower radioactivity than the control foetuses from rats injected with "°I (P<0.01) . The maternal blood, liver and thyroid gland of the venom-treated animals showed significantly lower

Fw 1 . TBRATOGENIC EPFECT OF A. amorcuxi vt3noM . Alizarin-stained cleared rat foetuses removed by Caesarean section on day 20 of gestation . (a) Abnormal foetus from mother treated with A . amoreuxi venom (0 .2 mg/kg i .p . daily on days 8 -12 of gestation) . (b) Control foetus from mother treated similarly but with saline. Note the missing, shonened, bent and deformed ribs, the missing ossification centres and the underdeveloped skull and vertebral column in the venom treated foetus . Note also the light stain in the venom-treated foetus resulting from diminished calcium deposition .

la2

FIG . 2 . EFFECT OF TREATMENT WITH PHENTOLAMINE AND TRIAMTERENE ON THE TERATOGENIC EFFECT OF A . QlItOI'CYX! VENOM,

Alizarin-stainod cleared rat foetuses removed by Caesarean section on day 20 of gestation. (a) Foetus removed from mother treated with ttiamtaene+ venom. (b) Foetus removed from mother treated with phentolamine+venom . The rats were injected with venom (0 .2 mg/kg i.p. daily) for 8 successive days (days 7 -14) . Trïamtaene (10 mg/rat orally daily) or phentolamine (1 mg/kg i.p . daily) was given 30 min before venom injection . Note the flattened and depressed skulls and the absence of ossification centres in the fore and hind limbs. Note also the stunted growth of the foetuses.

FIG . 3 . THE EFFECT' OF A . RIItOIplLY% VENOM ON 71~ PELVIC BLOOD VESSELS OF PREGNANT AND NONPREGNANT RATS.

A. amor~euri venom (0 .2 mg/kg i.p .) was injected daily on day g-12 of gestation. Control nonpregnant rats were similarly treated. Aü rats were killed on day 20 of gestation. Note the marked congestion of the pelvic blood vessels and the large haematoma behind the rectum in the area of the iliac, hypogastric and uterine arteries in the pregnant rats (a and c) as compared to control rats N).

Teratogenicity of Scorpion Venom TABLE

ConVol (6)

3.

183

EFFECT OF A . amOIIC1lXl VENOM ON BLOOD GLUCOSE AND PLASMA SODIUM, POTASSIUM. CALCIUM AND ALKALINE PHOSPHATASB OF PREGNANT RATS

Glucose Sodium mgt meq/1 87 141

Potassium meq/1 4.3

Calcium Alkaline phosphatase mg4b units/ml 9 .1 246

3 83

0.1 3.6 "

0.3 8.9

t

t

t

1

t

23 Vrnom-treated, 168" t t 1 t t 8 suocessive days 0.2 (days 7-14) (5) 3 5 0.2 13 87 141 3.9 " 8 .9 Phentolamine + 136" t t t t t vrnom-treated (days 7-14) (S) 7 7 0.2 0.2 20 Triamterrne + 72 " 142 3.6 " 8.6 122" venom treated t t t t t 3 4 0.2 0.3 18 (days 7 -14) (6) Figures in parentheses indicate number of rats in each group. Blood samples withdrawn on day 20 of gestation. Vrnom injected i.p . (0 .2 mg/kg) daily. Phrntolamine (1 mg/kg i.p.) injected 30 min before vrnom. Triamterrne (10 mg/rat orally) givrn 30 min before venom. "Values significantly different (P<0 .03) from the control group. TABLE KILLED

5 143

4. TISSUE DISTRIBUTION OF "~I-LABELLED A . aIIJOIICYX! VENOM AND' = 'I IN PREGNANT RATS 2 hr FOLLOWING THE LP .INJEGTION OF 1 mg/kg OF LABELLED VENOM OR'~~I (SPECffIC AC'r1VrfY 1 .2 NCi per mg) Radioactivity, counts/min per ml blood or organ Blood Foetuses Liver Kidney 37341322 3001 11 121061473 32001118 (1 .210 .1) (0 .1310.03) (3 .3910.48) (0 .ß9t0.1)

1

"°I-treated (day 11 of gestation) Vrnom-treated 1378173" 17819' 64871405 " 2348611263 " (1 .210 .1) (0 .1310.03) (3 .7110.19) (11 .610 .6) (day 10 of gestation) 33831772 " Venom-treated 9ß2f218" 158139" 1223213260" (1 .7810.4) (6.4310.58) (day 11 of (0.2610.05) (0 .0810 .02) gestation) 8571164' 746011000" Vrnom-treated 19291432 " 1955111971' (day 20 of (0.8910.13) (0.3310.06) (2 .9810.4) (7 .810 .8) gestation) Figures in parentheses are percentages of the total absorbed dose. Values are the average obtained from 6 animals in each group. "Significantly differrnt (P<0.03) from the "'I-treated control group.

S.E .M. Thyroid 1661011747 (3 .3310.36) 3l64ß62" (1 .5710 .18) 3098t2ß8" (1 .6310.15) 30381260 " (1 .210.1)

radioactivity than the control rats. On the other hand, the kidneys of the venom treated animals showed significantly higher radioactivity than the control rats . A similar distribution of radioactivity was found in the maternal tissues of rats at day 20 of gestation. The foetuses, however, had more radioactivity than the controls (Table 4). Radioactivity in the placenta and amniotic fluid removed from the rats at day 20 of gestation was 917 t 127 counts/min per placenta and 207 t 67 counts/min per ml amniotic fluid, respectively . DISCUSSION

A higher foetal resorption rate occurred when the scorpion venom was injected twice daily on days 9 and 10 of gestation and once on day 11 than when the same quantity of the

I86

M. ISMAIL, A. C . ELLISON and A. K . TILMISANY

venom was injected daily for 5 successive days starting from day 8 of gestation. This shows that days 9 -11 of gestation are probably critical in the action of the venom, Vertebral and ossification defects were observed in many of the viable foetuses obtained from mothers treated with scorpion venom. The type of the skeletal lesions and the type of vulnerability suggests a series of biochemical reactions occurring in the foetus and related to chondrification. More than one possibility could be presented to explain the nature and mechanism of the venom-induced teratogenesis . The most plausible one seems to be associated with the effect of the venom on carbohydrate metabolism (ISMAIL et al ., 1977, 1980). Embryonic bone is derived by oxidative pathways from mesenchymal and ectodermal differentiation. Mesoderm differentiation is dependent on succinic oxidase and folic acid, while ectoderm differentiation is dependent on glucose and cytochrome oxidase, with both systems dependent upon adequate oxygen saturation (DUFFEY and EBERT, 1957). A variety of agents affecting the cytochrome oxidase and succinic oxidase systems (folic acid, glucose and oxygen tension) have been reported to cause various skeletal abnormalities (RUNNER and DAGG, 1960) . As scorpion venoms have been reported to inhibit succinic dehydrogenase (MOUSTAFA et al., 1974), deplete liver and muscle glycogen (MOHAMED et al ., 1972 ; EL-ASMAR et al., 1974) and cause pronounced hyperglycemia (MOHAMED et al ., 1972 ; EL-ASMAR et al., 1974; ISMAIL et al., 1977, 1980), it seems possible that the teratogenic effect of the venom reported in the present work is linked with one or more of these actions. In addition, respiratory acidosis has been shown to occur in rabbits treated with A, amoreuxi venom (Ismail, Hadidy and Sarraj, unpublished data), indirectly reflecting reduced oxygen tension . It is also possible that the large haematoma observed in the treated rats and involving the area of the iliac, hypogastric and uterine arteries could contribute to uterine ischemia . The suggested effect on chondrification presupposes a specific chain of biochemical effects occurring in the foetus, although changes in the acid - base balance, as well as adverse pharmacological events, were elicited only in the maternal animals. Our working hypothesis is that abnormal carbohydrate metabolism in combination with an initial hypergalemia and subsequent hypogalemia is primarily responsible for the osteogenic malformations noted. In the present study, treatment with scorpion venom caused absence or delay in calcification of the ossification centers at days 19 and 20 of gestation, along with hypogycemia, hypoglycemia and a marked fall in serum alkaline phosphatase. This suggests that the actual effect was induced on the mesenchymal anlagen in the earlier stages of chondrification. Previous studies with A . amoreuxi venom in rabbits (ISMAIL et al., 1977, 1980) showed that the venom caused significant hyperglycemia, starting shortly after injection and lasting for more than 6 hr . This was associated with decreased glucose6-phosphate dehydrogenase and lactic dehydrogenase activity (ISMAIL et al., 1977). The venom also caused hypergycemia, hyperphosphatemia and respiratory acidosis (Ismail, Hadidy and Sarraj, unpublished data) along with a marked discharge of tissue and medullary catecholamines (GHAZAL et al., 1975) . As glucose concentration is markedly above control levels for a long time after venom injection, it seems possible that phosphorylated glucose in the hypertrophied cartilage may be decreased as a substrate for alkaline phosphatase in some later stage of chondrification. Support for this view comes from the marked increase in plasma phosphate, which suggests that the venom may interfere with the process of phosphorylation . Decreased intestinal absorption of calcium might be an additional factor in the observed hyperphosphataemia and consequently the

Teratogenicity of Scorpion Venom

18 7

reported ossification defects. A decreased intestinal absorption of calcium was reported for the venom from the scorpion Buthus minax (ISMAIL et al., 1978). The possibility that insulin, released as a consequence of the marked hyperglycemia, acts as the teratogenic agent is excluded for three reasons. Firstly, it was shown that the venom produces its hyperglycemic effect partly through the release of tissue and medullary catecholamines, which in turn inhibit insulin secretion from the ß-cells of the pancreatic islets via a-receptor stimulation (ISMAIL et al., 1977). Secondly, treatment of the rats with phentolamine, which blocks the inhibition of insulin release by catecholamines (ÀSHMORE, 1970), did not prevent the venom-induced ossification defects, although it prevented some vertebral and rib defects. Thirdly, although insulin has a direct effect on chondrification in chicken and mice (ZWELLING, 1959; RUNNER and DAGG, 1960; LANDAUER aril BLISS, 1946), no such correlation was found in the case Of rats (ELLISON Snd MAREN, 1972). Treatment with phentolamine or tolazoline significantly reduced, but did not block, the venom-induced hyperglycemia, however a slight delayed hyperglycemic response was ascribed to stimulated lipolysis and gluconeogenesis (ISMAIL et al., 1977) . In the present study, phentolamine reduced the foetal resorption rate caused by the venom, but had only a slight effect on chondrification or foetal weight loss . The hyperglycemic response might be responsible for the foetal mortality, but alone was not a decisive factor in the chondriflcation process. It has been reported (PRESSMAN ând LARDY, 1952; BLOND änd WHITTAM, 1965) that potassium regulates oxidative phosphorylation and cellular respiration and that this regulation is maintained by the Na+/K' ratio of the cell . Increased Na+ concentration or decreased K' concentration inhibit these reactions, while potassium deficiency causes increased glycogenolysis (BARTELHEIMER et al., 1967). Scorpion venom was shown by ADAM and WEISS (1966) to cause a slow depolarization of the excitable membrane of nerve and muscle due to an increase in sodium permeability and a delayed inactivation of this permeability . Also, WIDMAN (1956) showed that repolarization may result from an efflux of intracellular potassium. A. amoreuxi venom caused a significant lowering of serum sodium and a significant elevation of serum potassium 45 - 75 min following injection into rabbits (Ismail, Hadidy and Sarraj, unpublished data). It was thought that triamterene might be effective in inhibiting the venom-induced teratogenicity by restoring the Na`/K` ratio back to normal . Treatment with triamterene reduced the foetal resorption rate and significantly prevented the effects of the venom on chondrification. However, marked stippling .was observed in the long bones and ribs, possibly due to increased mobilization of ionized calcium in the foetus . It was shown that small doses of scorpion venom injected into rats over a period of several days caused a decrease in serum calcium and phosphorus ((EL-ASMER et al., 1975), while higher doses inhibited intestinal absorption of calcium and elevated serum phosphorus (ISMAIL et al., 1978). Treatment of rats with scorpion venom over a longer period of time and starting at an earlier time of gestation (days 7 -14) caused total foetal resorption. This might be due to inhibition of histamine formation, as scorpion venom was shown to inhibit the specific histidine decarboxylase in the rat (ISMAIL and OSMAN, 1973). It is known that implantation of the fertilized ovum in the rat occurs at day 6 of gestation (CHRISTIE, 1964) and that histamine is essential for nidation, acting as a mediator for cestrogens (SHELESNYAK, 1959, 1960 ; MARCUS et al., 1964; FERRANDO and NALHANDOV, 1968). It was also reported that the process of histamine formation is connected with the intrauterine growth of the rat foetus (KAHLSON et al., 1958, 1959; KAHLSON, 1960) and'

188

M . ISMAIL, A . C . ELLISON and A. K . TILMISANY

that inhibition of L-histamne decarboxylase arrests foetal development in the rat (ICAHLSON and ROSENGREN, 1971) . The teratogenic effect of the venom appears to be the result of its metabolic effect_ and action on maternal body electrolytes, rather than to a direct effect on the foetuses . Evidence for this comes from measurement of radioactivity in the foetuses at day 10 and 11 of gestation and in the foetuses, placenta and amniotic fluid at day 20 . Only a small fraction of the injected dose (0 .08 -0.33%) was detected in these sites . The distribution of the venom in the uterus and the pattern of its distribution in other organs is similar to that reported for Leiurus quinquestriatus venom in the rat and A. amor~euxi venom in the rabbit (ISMAIL et al ., 1974b, 1980) .

Acknowledgement - This work was supported by grant PHRCI from the Research Centre, College of Pharmacy, King Saud University.

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