Bull. Environ. Contam. Toxicol. (1991) 47:641-645 9 1991 Springer-Verlag New York Inc.
~Environmental
~I;::.airily_m ina.tion ~ana ]ox,coaogy
Effect of a Single Oral Dose of Metrifonate on Human Plasma Cholinesterase Levels Charles F. B. Nhachi, 1 Wilbert Murambiwa, 1 0 s s y J. Kasilo, 2 Lovemore Gwanzura, 3 and Peter Mason 3 ~Clinical Pharmacology, 2pharmacy and 3Medical Microbiology, School of Medicine, University of Zimbabwe, P.O. Box A178, Avondale Harare, Zimbabwe Clinically, m e t r i f o n a t e or t r i c h l o r f o n 2,2,2 - trichloroethyl d i m e t h y l phosphate), or D i p t e r e x is an organop h o s p h o r u s compound. C o n s e q u e n t l y m e t r i f o n a t e exhibits most of the pharmacological and toxicological characteristics of the organophosphorus group of compounds, i.e.characteristically it inhibits the a c t i v i t y of a c e t y l c h o l i n e s t e r a s e . Significant exposure to m e t r i f o n a t e results in the well known and well studied signs and symptoms of o r g a n o p h o s h a t e p o i s o n i n g (Goulding, 1981). While occupational exposure to organophosphates is common, (mostly in a g r i c u l t u r e as insecticides), (Lu, 1984), e x p o s u r e to m e t r i f o n a t e is m o s t l y through its therapeutic use in the control and treatment of schistosoma haematobium infection, (el Kholy, 1984; Snellen, 1981). In Zimbabwe, metrifonate is used to treat bilharzia. It is a d m i n i s t e r e d as a single oral dose of 7.5 - 10mg per k i l o g r a m body w e i g h t (Ministry of Health, 1989). The p r e s e n t study was carried out to m e a s u r e the d u r a t i o n of i n h i b i t i o n and s i g n i f i c a n c e of r e c o v e r y of p l a s m a c h o l i n e s t e r a s e activity in S c h i s t o s o m a h a e m a t o b i u m i n f e c t e d school c h i l d r e n after a single oral dose of m e t r i f o n a t e . M E T H O D S AND M A T E R I A L S The study was carried out at a p r i m a r y school in an area that is endemic w i t h Schistosoma haematobium, in the north - east part of the country. A single oral (tablets) dose of m e t r i f o n a t e , 10mg/kg body w e i g h t was a d m i n i s t e r e d to g r a d e 3 school children, (24 girls and 25 boys, w i t h an average age of 11.4 + 0.5 years, average w e i g h t of 24.4 + 3.3 kilograms). Blood plasma c h o l i n e s t e r a s e levels w e r e assayed i m m e d i a t e l y before a d m i n i s t r a t i o n of the drug and 3 times i.e. 2 days, Send R e p r i n t s requests above address.
to
Charles
641
F.B.
Nhachi
at
the
5 days and 30 days of Edison, 1955.
after
treatment,
using
the
method
Exposure to o r g a n o p h o s p h a t e s can be determined by o b s e r v i n g the levels of normal c h o l i n e s t e r a s e a c t i v i t y in the blood. In the p r e s e n t study, m e a s u r e m e n t of blood cholinesterase activity was used as a q u i c k and accurate way of screening school children for p o s s i b l e m e t r i f o n a t e exposure. The s t a n d a r d l o v i b o n d c o m p a r a t o r disc 5.30 (Limperas and Rama), uses normal enzyme a c t i v i t y in steps of 12.5 per cent and is c a l i b r a t e d for use w i t h 2.5 m m cells. Further discrimination of + 5 per cent can be a c h i e v e d by a s s e s s i n g the d i r e c t i o n of the colour d i f f e r e n c e b e t w e e n the m o s t c l o s e l y m a t c h i n g colours. The reagents w e r e t e s t e d by m i x i n g 0.5 ml of i n d i c a t o r (Bromo T h y m o l Blue), 0.i ml of f i n g e r p r i c k blood form an u n e x p o s e d 'control' subject, and 0.5 ml of s u b s t r a t e (actylcholine perchlorate) solution in a test tube. The w e l l - m i x e d contents w e r e p l a c e d in a 2.5 m m cell in the r i g h t - h a n d compartment of the c o m p a r a t o r and c o m p a r e d w i t h a test s o l u t i o n by r e v o l v i n g the disc until a close colour match with the test solution is obtained. Control blood was obtained from the researchers. These researchers had no history of exposure to organophosphates. F r o m each subject, 0.1 ml of b l o o d (finger-prick) was p i p e t t e d into a r e a c t i o n tube w i t h the indicator. Temperature, taken in the shade was noted and 0.5 ml of s u b s t r a t e was added to the r e a c t i o n tube. E a c h tube was allowed to stand for e x a c t l y the time c o r r e s p o n d i n g to the shade t e m p e r a t u r e shown in the Time - Temperature Table provided with the kit. The r e a c t i o n tube c o n t e n t s w e r e then i m m e d i a t e l y t r a n s f e r r e d to the 2.5 m m sample cell, w h i c h was p l a c e d in the r i g h t - h a n d compartment of the c o m p a r a t o r and m a t c h e d a g a i n s t the s t a n d a r d s in the disc. The results are c o n s i s t e n t w i t h those o b t a i n e d by the e l e c t r o m e t r i c m e t h o d of M i c h e l (1949). The c h o l i n e s t e r a s e a c t i v i t y is e x p r e s s e d as a p e r c e n t a g e of the a c t i v i t y in normal blood and less than 75 p e r c e n t of the n o r m a l a c t i v i t y was taken as a s i g n i f i c a n t d e c r e a s e (Edson, 1955). RESULTS
AND DISCUSSION
The use of a single oral dose of m e t r i f o n a t e in the treatment of Schistosoma haematobium infection is efficacous and w i d e l y used, (Aden-Abdi et al, 1987; F e l d m e i e r et al, 1982; d r u i l h e et al 1981; M i n i s t r y of Health, 1989). The
results
of
our
study 642
confirm
the
efficacy
of
Table
i.
E f f e c t of a s i n g l e o r a l d o s e of m e t r i f o n a t e on C h o l i n e s t a r a s e l e v e l s in P r i m a r y s c h o o l children.
Cholinestarase levels A c t i v i t y as % of B a s e l i n e Line Activity
30 41 51 61 71 81 91
- 40 - 50 - 60 - 70 - 80 - 90 -100
Number
of S u b j e c t s
Pretreatment "Controls" 0 Days
0 0 0 0 19 (39%) 16 (33%) 14 (28%)
Total
number
Table
2.
of s t u d e n t s
2 Days after treatment
0 5 (10%) 0 0 18 (37%) 18 (37%) 8 (16%) 2 (4%)
30 D a y s after treat ment
1 (2%) 3 (6%) 0 17(35%) 19(39%) 7 (14%) 2 ( 4%)
0 0 0 0 0 21(42.1%) 28(57.9%)
N = 49
R e l a t i o n s h i p b e t w e e n the n u m b e r of s u b j e c t s with cholinesterase levels activity s i g n i f i c a n t l y i n h i b i t e d a n d the n u m b e r of days after treatment.
Pretreatment '0' D a y s treatment
2 Days after treatment
5 Days after treatment
30 D a y s after treatment
21
0
43%
0
N u m b e r of subjects with cholinesterase a c t i v i t y < 70% of c o n t r o l s
0
23
% of s u b j e c t s w i t h < 70% c h o l i nesterase activity
0
47%
Where
5 Days after treatment
N = 49
metrifonate in inhibiting cholinesterase activity, (table 1 and 2). Cholinesterase a c t i v i t y of 43% of the s u b j e c t s w a s d e t e r m i n e d to be less t h a n 70% of normal for a p e r i o d of at l e a s t 5 days. T h e last 643
c h o l i n e s t e r a s e assay was done 30 days after treatment, to reduce the f r e q u e n c y of the trauma of finger-prick. However our results indicated that at most, by the end of month, cholinesterase a c t i v i t y had r e c o v e r e d back to normal levels in all the subjects. Similar results have been shown by A d e n - A b d i et al 1987. In view of the fact that m e t r i f o n a t e m a y be toxic, (Csik et al, 1986; Hu et al, 1986), and based on the results from this study, we recommend that after t h e r a p e u t i c t r e a t m e n t w i t h m e t r i f o n a t e subjects should avoid o c c u p a t i o n a l e x p o s u r e to o r g a n o p h o s p h a t e s since this might exacebate the toxic effects of these compounds. Acknowledgements. Our thanks go to the p r i m a r y s c h o o l c h i l d r e n for their co-operation. F u n d i n g of the " A g r o c h e m i c a l s H e a l t h Hazards" p r o j e c t is by the Swidish A g e n c y for R e s e a r c h C o - o p e r a t i o n w i t h D e v e l o p ping Countries, (SAREC). REFERENCES A d e n - a b d i Y, G u s t a f f s s o n LL, Elmi SA (1987) A s i m p l i f i e d dosage s c h e d u l e of m e t r i f o n a t e in the t r e a t m e n t of s c h i s t o s o m a h a e m a t o b i u m i n f e c t i o n in Somalia. Eur J Clin P h a r m a c o l 32; 432 - 441. Csik V, M o t i k a D, M a r o s i GY (1986) Delayed neuropathy after t r i c h l o r f o n intoxication. Neurosurg P s y c h i a t r y 49; 222 - 222. Druilhe P, B o u r d i l l o n F, F r o m e n t A, K y e l e m JM (1981) C o n t r o l of u r i n a r y s c h i s t o s o m i a s i s by 3 annual courses of m e t r i f o n a t e . Ann Soc Belg Med T r o p 61; 99 - 109. Edson EF (1955) E m e r g e n c i e s in g e n e r a l practice. A g r i c u l t u r a l pesticides. Br. M e d J; i: 841 - 844. e l - K h o l y A, Boutros S, T a m a r a F, W a r r e n KS, M a h m o u n d A A (1984) The effect of a single dose of m e t r i f o n a t e on S c h i s t o s o m a h a e m a t o b i u m i n f e c t i o n in E g y p t i a n school children. A m J Tro M e d Hyg 33; 1170 - 1172. F e l d m e i e r H, D o e h r i n g E, D a f f a l l a AA, Omer AH, D e i t r i c h M (1982) E f f i c a c y of m e t r i f o n a t e in u r i n a r y s c h i s t o s o m i a s i s , c o m p a r i s o n of r e d u c t i o n of s c h i s t o s o m a h a e m a t o b i u m and m a n s o n i eggs. Am J T r o p Med Hyg 31: 1188 - 1194. G o u l d i n g R (1981) P o i s o n i n g due to pesticides. In Vale, JA and M e r e d i t h TJ (ed) P o i s o n i n g d i a g n o s i s and treatment, Update Books; London: Dordrect, Boston, PP 144 - 145. L i m p e r a s G, R a n t a KE (1953) The rapid field d e t e r m i n a tion of c h o l i n e s t e r a s e . Science 117: 453 - 457. Hu XH, Lu TP, Xue SZ, L i n y XY, Gu XQ (1986) T o x i c i t y of Dipterex, a field study. Br J Ind M e d 4 3 : 4 1 4 - 419. M i c h e l HD (1949). The e l e c t r o m e t r i c d e t e r m i n a t i o n of 644
c n o l i n e s t e r a s e . J. Lab Clin Med: 34, 1954 - 1956. M i n i s t r y of Health, G o v e r n a m e n t of Zimbabwe (1989). The e s s e n t i a l drugs list for Zimbabwe pp 55. Snellen WM (1981) T h e r a p e u t i c p r o p e r t i e s of m e t r i f o n a he. A c t a P h a r m a c o l T o x i c o l 49: Suppl 5 : 1 1 4 - 117. R e c e i v e d O c t o b e r 18, 1 9 9 0 ; a c c e p t e d A p r i l 5, 1991.
645
~Environmental
~I;::.airily_m ina.tion ~ana ]ox,coaogy
Effect of a Single Oral Dose of Metrifonate on Human Plasma Cholinesterase Levels Charles F. B. Nhachi, 1 Wilbert Murambiwa, 1 0 s s y J. Kasilo, 2 Lovemore Gwanzura, 3 and Peter Mason 3 ~Clinical Pharmacology, 2pharmacy and 3Medical Microbiology, School of Medicine, University of Zimbabwe, P.O. Box A178, Avondale Harare, Zimbabwe Clinically, m e t r i f o n a t e or t r i c h l o r f o n 2,2,2 - trichloroethyl d i m e t h y l phosphate), or D i p t e r e x is an organop h o s p h o r u s compound. C o n s e q u e n t l y m e t r i f o n a t e exhibits most of the pharmacological and toxicological characteristics of the organophosphorus group of compounds, i.e.characteristically it inhibits the a c t i v i t y of a c e t y l c h o l i n e s t e r a s e . Significant exposure to m e t r i f o n a t e results in the well known and well studied signs and symptoms of o r g a n o p h o s h a t e p o i s o n i n g (Goulding, 1981). While occupational exposure to organophosphates is common, (mostly in a g r i c u l t u r e as insecticides), (Lu, 1984), e x p o s u r e to m e t r i f o n a t e is m o s t l y through its therapeutic use in the control and treatment of schistosoma haematobium infection, (el Kholy, 1984; Snellen, 1981). In Zimbabwe, metrifonate is used to treat bilharzia. It is a d m i n i s t e r e d as a single oral dose of 7.5 - 10mg per k i l o g r a m body w e i g h t (Ministry of Health, 1989). The p r e s e n t study was carried out to m e a s u r e the d u r a t i o n of i n h i b i t i o n and s i g n i f i c a n c e of r e c o v e r y of p l a s m a c h o l i n e s t e r a s e activity in S c h i s t o s o m a h a e m a t o b i u m i n f e c t e d school c h i l d r e n after a single oral dose of m e t r i f o n a t e . M E T H O D S AND M A T E R I A L S The study was carried out at a p r i m a r y school in an area that is endemic w i t h Schistosoma haematobium, in the north - east part of the country. A single oral (tablets) dose of m e t r i f o n a t e , 10mg/kg body w e i g h t was a d m i n i s t e r e d to g r a d e 3 school children, (24 girls and 25 boys, w i t h an average age of 11.4 + 0.5 years, average w e i g h t of 24.4 + 3.3 kilograms). Blood plasma c h o l i n e s t e r a s e levels w e r e assayed i m m e d i a t e l y before a d m i n i s t r a t i o n of the drug and 3 times i.e. 2 days, Send R e p r i n t s requests above address.
to
Charles
641
F.B.
Nhachi
at
the
5 days and 30 days of Edison, 1955.
after
treatment,
using
the
method
Exposure to o r g a n o p h o s p h a t e s can be determined by o b s e r v i n g the levels of normal c h o l i n e s t e r a s e a c t i v i t y in the blood. In the p r e s e n t study, m e a s u r e m e n t of blood cholinesterase activity was used as a q u i c k and accurate way of screening school children for p o s s i b l e m e t r i f o n a t e exposure. The s t a n d a r d l o v i b o n d c o m p a r a t o r disc 5.30 (Limperas and Rama), uses normal enzyme a c t i v i t y in steps of 12.5 per cent and is c a l i b r a t e d for use w i t h 2.5 m m cells. Further discrimination of + 5 per cent can be a c h i e v e d by a s s e s s i n g the d i r e c t i o n of the colour d i f f e r e n c e b e t w e e n the m o s t c l o s e l y m a t c h i n g colours. The reagents w e r e t e s t e d by m i x i n g 0.5 ml of i n d i c a t o r (Bromo T h y m o l Blue), 0.i ml of f i n g e r p r i c k blood form an u n e x p o s e d 'control' subject, and 0.5 ml of s u b s t r a t e (actylcholine perchlorate) solution in a test tube. The w e l l - m i x e d contents w e r e p l a c e d in a 2.5 m m cell in the r i g h t - h a n d compartment of the c o m p a r a t o r and c o m p a r e d w i t h a test s o l u t i o n by r e v o l v i n g the disc until a close colour match with the test solution is obtained. Control blood was obtained from the researchers. These researchers had no history of exposure to organophosphates. F r o m each subject, 0.1 ml of b l o o d (finger-prick) was p i p e t t e d into a r e a c t i o n tube w i t h the indicator. Temperature, taken in the shade was noted and 0.5 ml of s u b s t r a t e was added to the r e a c t i o n tube. E a c h tube was allowed to stand for e x a c t l y the time c o r r e s p o n d i n g to the shade t e m p e r a t u r e shown in the Time - Temperature Table provided with the kit. The r e a c t i o n tube c o n t e n t s w e r e then i m m e d i a t e l y t r a n s f e r r e d to the 2.5 m m sample cell, w h i c h was p l a c e d in the r i g h t - h a n d compartment of the c o m p a r a t o r and m a t c h e d a g a i n s t the s t a n d a r d s in the disc. The results are c o n s i s t e n t w i t h those o b t a i n e d by the e l e c t r o m e t r i c m e t h o d of M i c h e l (1949). The c h o l i n e s t e r a s e a c t i v i t y is e x p r e s s e d as a p e r c e n t a g e of the a c t i v i t y in normal blood and less than 75 p e r c e n t of the n o r m a l a c t i v i t y was taken as a s i g n i f i c a n t d e c r e a s e (Edson, 1955). RESULTS
AND DISCUSSION
The use of a single oral dose of m e t r i f o n a t e in the treatment of Schistosoma haematobium infection is efficacous and w i d e l y used, (Aden-Abdi et al, 1987; F e l d m e i e r et al, 1982; d r u i l h e et al 1981; M i n i s t r y of Health, 1989). The
results
of
our
study 642
confirm
the
efficacy
of
Table
i.
E f f e c t of a s i n g l e o r a l d o s e of m e t r i f o n a t e on C h o l i n e s t a r a s e l e v e l s in P r i m a r y s c h o o l children.
Cholinestarase levels A c t i v i t y as % of B a s e l i n e Line Activity
30 41 51 61 71 81 91
- 40 - 50 - 60 - 70 - 80 - 90 -100
Number
of S u b j e c t s
Pretreatment "Controls" 0 Days
0 0 0 0 19 (39%) 16 (33%) 14 (28%)
Total
number
Table
2.
of s t u d e n t s
2 Days after treatment
0 5 (10%) 0 0 18 (37%) 18 (37%) 8 (16%) 2 (4%)
30 D a y s after treat ment
1 (2%) 3 (6%) 0 17(35%) 19(39%) 7 (14%) 2 ( 4%)
0 0 0 0 0 21(42.1%) 28(57.9%)
N = 49
R e l a t i o n s h i p b e t w e e n the n u m b e r of s u b j e c t s with cholinesterase levels activity s i g n i f i c a n t l y i n h i b i t e d a n d the n u m b e r of days after treatment.
Pretreatment '0' D a y s treatment
2 Days after treatment
5 Days after treatment
30 D a y s after treatment
21
0
43%
0
N u m b e r of subjects with cholinesterase a c t i v i t y < 70% of c o n t r o l s
0
23
% of s u b j e c t s w i t h < 70% c h o l i nesterase activity
0
47%
Where
5 Days after treatment
N = 49
metrifonate in inhibiting cholinesterase activity, (table 1 and 2). Cholinesterase a c t i v i t y of 43% of the s u b j e c t s w a s d e t e r m i n e d to be less t h a n 70% of normal for a p e r i o d of at l e a s t 5 days. T h e last 643
c h o l i n e s t e r a s e assay was done 30 days after treatment, to reduce the f r e q u e n c y of the trauma of finger-prick. However our results indicated that at most, by the end of month, cholinesterase a c t i v i t y had r e c o v e r e d back to normal levels in all the subjects. Similar results have been shown by A d e n - A b d i et al 1987. In view of the fact that m e t r i f o n a t e m a y be toxic, (Csik et al, 1986; Hu et al, 1986), and based on the results from this study, we recommend that after t h e r a p e u t i c t r e a t m e n t w i t h m e t r i f o n a t e subjects should avoid o c c u p a t i o n a l e x p o s u r e to o r g a n o p h o s p h a t e s since this might exacebate the toxic effects of these compounds. Acknowledgements. Our thanks go to the p r i m a r y s c h o o l c h i l d r e n for their co-operation. F u n d i n g of the " A g r o c h e m i c a l s H e a l t h Hazards" p r o j e c t is by the Swidish A g e n c y for R e s e a r c h C o - o p e r a t i o n w i t h D e v e l o p ping Countries, (SAREC). REFERENCES A d e n - a b d i Y, G u s t a f f s s o n LL, Elmi SA (1987) A s i m p l i f i e d dosage s c h e d u l e of m e t r i f o n a t e in the t r e a t m e n t of s c h i s t o s o m a h a e m a t o b i u m i n f e c t i o n in Somalia. Eur J Clin P h a r m a c o l 32; 432 - 441. Csik V, M o t i k a D, M a r o s i GY (1986) Delayed neuropathy after t r i c h l o r f o n intoxication. Neurosurg P s y c h i a t r y 49; 222 - 222. Druilhe P, B o u r d i l l o n F, F r o m e n t A, K y e l e m JM (1981) C o n t r o l of u r i n a r y s c h i s t o s o m i a s i s by 3 annual courses of m e t r i f o n a t e . Ann Soc Belg Med T r o p 61; 99 - 109. Edson EF (1955) E m e r g e n c i e s in g e n e r a l practice. A g r i c u l t u r a l pesticides. Br. M e d J; i: 841 - 844. e l - K h o l y A, Boutros S, T a m a r a F, W a r r e n KS, M a h m o u n d A A (1984) The effect of a single dose of m e t r i f o n a t e on S c h i s t o s o m a h a e m a t o b i u m i n f e c t i o n in E g y p t i a n school children. A m J Tro M e d Hyg 33; 1170 - 1172. F e l d m e i e r H, D o e h r i n g E, D a f f a l l a AA, Omer AH, D e i t r i c h M (1982) E f f i c a c y of m e t r i f o n a t e in u r i n a r y s c h i s t o s o m i a s i s , c o m p a r i s o n of r e d u c t i o n of s c h i s t o s o m a h a e m a t o b i u m and m a n s o n i eggs. Am J T r o p Med Hyg 31: 1188 - 1194. G o u l d i n g R (1981) P o i s o n i n g due to pesticides. In Vale, JA and M e r e d i t h TJ (ed) P o i s o n i n g d i a g n o s i s and treatment, Update Books; London: Dordrect, Boston, PP 144 - 145. L i m p e r a s G, R a n t a KE (1953) The rapid field d e t e r m i n a tion of c h o l i n e s t e r a s e . Science 117: 453 - 457. Hu XH, Lu TP, Xue SZ, L i n y XY, Gu XQ (1986) T o x i c i t y of Dipterex, a field study. Br J Ind M e d 4 3 : 4 1 4 - 419. M i c h e l HD (1949). The e l e c t r o m e t r i c d e t e r m i n a t i o n of 644
c n o l i n e s t e r a s e . J. Lab Clin Med: 34, 1954 - 1956. M i n i s t r y of Health, G o v e r n a m e n t of Zimbabwe (1989). The e s s e n t i a l drugs list for Zimbabwe pp 55. Snellen WM (1981) T h e r a p e u t i c p r o p e r t i e s of m e t r i f o n a he. A c t a P h a r m a c o l T o x i c o l 49: Suppl 5 : 1 1 4 - 117. R e c e i v e d O c t o b e r 18, 1 9 9 0 ; a c c e p t e d A p r i l 5, 1991.
645
