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Response of blood and brain cholinesterase to dermal exposure of bromophos in the rat
Toxicology, 48 (1988) 1 9 9 - 2 0 8 Elsevier Scientific Publishers Ireland Ltd.
R E S P O N S E O F B L O O D A N D B R A I N C H O L I N E S T E R A S E TO D E R M A L E X P O S U R E O F B R O M O P H O S IN T H E R A T
T. SHIVANANDAPPA*,PIUS JOSEPH and M.K. KRISHNAKUMARI Toxicology Uni~ Infestation Control and Protectants Discipline, Central Food Technological Research Institute, Mysore - 570 013 (India)
(Received April 2nd, 1987} (Accepted September 13th, 1987) SUMMARY
Response of cholinesterase to dermal exposure of acute, single and multiple doses of Bromophos in the female rat has been studied. Doseresponse studies (50--4000 mg/kg body weight, 24 h exposure) showed that plasma cholinesterase was most sensitive to inhibition in vivo, followed by the brain and erythrocyte acetylcholinesterase. The ID5o values for the in vivo cholinesterase inhibition were 10.1, 576.1 and 1938.0 mg/kg body weight for the plasma, brain and erythrocytes, respectively. In time-course studies after a single sublethal dose of 1000 mg/kg body weight (24 h) of Bromophos, the serum and brain cholinesterase were rapidly inhibited reaching a maximum at 16 h. Recovery was complete in the case of serum at 14 days post-exposure, whereas the brain enzyme was not fully recovered at 21 days. In a subacute study, daily dermal application of 50 mg/kg body weight of Bromophos for 5 and 10 days, resulted in high inhibition of the serum cholinesterase and brain acetylcholinesterase, the former being more marked which was reversible after 10 or 15 days of post-exposure period. Very low levels of dermal exposure of Bromophos (10--50 mg/kg body weight) for 17 days caused pronounced depression of serum cholinesterase which completely recovered in 15 days after cessation of exposure suggesting that the serum cholinesterase could serve as the most sensitive diagnostic indicator of Bromophos exposure. w o r d s : Bromophos; O,O-Dimethyl-O(2,5-dichloro-4-bromophenyl}-phosphorothioate; Pseudocholinesterase; Acetylcholinesterase; Brain; Erythrocytes; Dermal toxicity
Key
*To whom all correspondence and reprint requests should be addressed. ACHE, acetylcholinesterase; Bromophos, [O,O-dimethyl-O-(2,5-dichloro-4bromophenyt) phosphorothioate]; pChE, pseudocholinesterase.
Abbreviations:
0300-483X/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
199
INTRODUCTION
Bromophos [O, O-dimethyl-O-(2,5-dichloro-4-bromophenyl)phosphorothioate] is a non-systemic organophosphorus insecticide widely used in crop protection, infestation control of stored grain, public health and on ticks and mites of domestic animals [1--4]. One distinctive feature of Bromophos, among organophosphorus insecticides, is its extremely low acute toxicity to mammals with an LD~0 of 3700--6100 mg/kg body weight in the rat and 3311 --6000 mg/kg body weight in mice [5--7]. It is quickly absorbed and excreted when ingested orally with a biological half-life of 14 h in the rat [20]. Bromophos undergoes metabolic activation and is an inhibitor of acetylcholinesterase in vivo in mammals [8,9]. Because of its very low acute toxicity, Bromophos has been specially recommended for the ectoparasite control of domestic animals [3]. Gaines [10] has reported that acute dermal LDs0 for Bromophos lies beyond 5000 mg/kg body weight to rats but there is no information on the response of cholinesterase to dermal exposure. Since Bromophos is a solid at room temperature with a negligible vapour pressure [11], its main route to occupational exposure to handlers and agricultural workers will be through percutaneous absorption. Hence, for an exposure risk assessment and monitoring, it is essential to understand the effects of Bromophos after dermal application. The present study was therefore undertaken to investigate the acute and subacute toxicity of Bromophos with reference to cholinesterase inhibition and recovery in the blood and brain. MATERIALS AND METHODS
A n i m a l s and t r e a t m e n t
Female adult albino rats of Wistar strain weighing 1 8 0 - 2 0 0 g were housed in individual cages, grouped into 4 rats/group and provided with food and water ad lib. The skin on the dorsal side behind the neck was shaved with an electrically operated hair clipper. The clipped area was 3 × 2 cm. Technical bromophos (93% pure) was synthesised in this Institute which was identical to the authentic Bromophos {Environmental Protection Agency, U.S.A.; and CelaMerck, Darmstadt, Germany) in terms of physical, chemical and insecticidal properties [12]. Bromophos {solid, white crystalline powder) was dissolved in ethyl acetate and applied evenly as a thin layer on the shaven skin surface. Control rats were treated with ethyl acetate alone. For acute dose-response studies, doses of Bromophos ranging from 50 to 4000 mg/kg body weight in a maximum of 0.5 ml of the carrier were applied and after 24 h of exposure, the animals were sacrificed. For time-course studies, a single acute sublethal dose of 1000 mg/kg body weight was applied, rats were killed at various time intervals of 4, 8, 16 and 24 h. For recovery studies groups of rats after 24 h of dermal exposure to Bromophos, the applied area of the skin was cleaned with alcohol and soapy water and the animals killed at 1, 2, 3, 6, 7, 10, 14 and 21 days. Two subacute studies were made: (i) daily dermal application of 50
200
mg/kg body weight of Bromophos in 0.2 ml of ethyl acetate for 5 and 10 days with a recovery period of 5, 10 and 15 days after the 10th application; and (ii) daily application of 10, 25 and 50 mg/kg body weight of Bromophos for 17 days and a recovery period of 15 days after the last application to monitor the serum cholinesterase as a diagnostic indicator of low level of exposure to Bromophos.
Sample preparation Sacrifice of animals was done after diethyl ether anaesthesia. For the separation of erythrocytes, blood was drawn in heparinised syringes, centrifuged at 1000 x g. and the plasma was collected; the sediment was washed twice with 0.9% saline and the erythrocytes were suspended in saline to the original blood volume. At the time of assay for acetylcholinesterase, an aliquot of the erythrocyte suspension was lysed with distilled water and used as the enzyme source. To obtain serum, blood was collected directly from the heart without any anticoagulant, allowed to clot and then centrifuged at 1000 x g. for 15 rain. Brains were removed, washed in saline and homogenised in 0.1 M ice-cold phosphate buffer (pH 7.4) using a Potter-Elvehjem motor-driven homogeniser, centrifuged at 1000 x g. and the supernatant was used for acetylcholinesterase assays.
Enzyme assays Pseudocholinesterase (EC 3.1.1.8 pChE) in the plasma or serum, and acetylcholinesterase (EC 3.1.1.7 ACHE) in the brain and erythrocytes were assayed by the method of Ellman et al. [13] with acetylthiocholine iodide (Merck, Darmstadt, F.R.G.) as the substrate by recording the change in absorbance every 30 s for 3 rain at 412 nm. The enzyme activity was calculated by the extinction coefficient (~:412 ~-~ 1.36 x 10~ M -1 cm-9 for the yellow anion, 5,dithio-2, nitrobenzoid acid [14], and expressed as pmol min -~ or percent of control.
Statistical analyses Differences between the treated and control groups were analysed by Student's' t-test [15]. Significance was assessed at 0.1 or 1% level (P < 0.001 or < 0.01L In vivo IDs0 were calculated from the dose-response data of cholinesterase inhibition by probit regression analysis [16]. RESULTS
Dose-response study There was no mortality up to the highest dosage (4000 mg/kg body weight) of Bromophos. However, mild symptoms appeared at 2000 mg/kg body weight and were well pronounced at 4000 mg/kg body weight. The symptoms were typically cholinergic such as lacrimation, salivation, urination and nasal discharge which were mild at 8 h of exposure, distinct at 24 h, lasted up to 48 h and by 72 h, the rats had completely recovered. Except in the highest dose, food intake was not affected.
201
• Plasma [ ] Brain [ ] Erythrocyte
100
8
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LtJ
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t
20
50
100
250
500
1000
2000
4000
Dose (mg/kg b.w.) Fig. 1. Dose-response of plasma cholinesterase, erythrocyte and brain acetylcholinesterase inhibition in female rats after a 24 h acute dermal exposure to Bromophos. Each value (% of control) is the Mean _+ S.E. (vertical bars) of 4 animals/group.
pChE in the plasma and, AChE in erythrocytes and brain showed dosedependent inhibition (Fig. 1). Marked inhibition (61o/o) of pChE was seen even at the lowest dosage and it was maximum (86O/o) at 2000 and 4000 mg/kg body weight. No significant inhibition of AChE in the brain was seen at the lowest dosage, but it was dose-dependent beyond 100 mg/kg body weight which ranged from 360/o to 67o/o (Fig. 1). It may be noted that inhibition did not increase beyond 2000 mg/kg body weight and the onset of symptoms was evident with the high AChE inhibition in the brain. Erythrocyte AChE was not affected at 50 and 100 mg/kg body weight but was inhibited beyond 250 TABLE I IN VIVO SENSITIVITY OF CHOLINESTERASE TO ACUTE DERMAL BROMOPHOS IN THE FEMALE RAT Enzyme source
IDa0*
Probit regression equation**
Plasma Brain Erythrocyte
10.1 567.1 1938.0
Y = 4.5155 + 0.4811 x Y = 2.0718 + 1.0634 x Y = 1.7792 + 0.0798 x
DOSES
OF
*The dose (mg/kg body weight for 24 h) required to produce 50% inhibition of cholinesterase in vivo. **Values were computed by probit regression analysis from the data shown in Fig. 1.
202
~-
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Fig. 2. Time course of inhibition of serum cholinesterase (pChE) and brain acetyl cholinesterase (ACHE) in female rats after a single acute dermal application of a sublethal dose (1000 mg/kg body weight) of Bromophos. Values (% of control) at each time interval are expressed as Mean +_ S.E. (vertical bars) of 4 animals. (See Methods for experimental details and Table II for control values of cholinesterase).
mg/kg body weight and the inhibition progressed beyond 2000 mg/kg body weight unlike the plasma and brain enzyme (Fig. 1). IDs0 values show vast differences of the cholinesterase of the plasma, brain, and e r y t h r o c y t e s in their susceptibility to inhibition in vivo by Bromophos, viz., 10.1, 567.1 and 1938 mg/kg body weight, respectively (Table I).
Time-course of cholinesterase inhibition and recovery Figure 2 depicts the time-course inhibition and recovery pattern of serum pChE and brain AChE after 24 h dermal exposure to a single sublethal dose (1000 mg/kg body weight) of Bromophos. The inhibition in both serum and brain could be seen as early as 4 h which progressed to reach a maximum at 16 h of exposure and it was unchanged up to 24 h. Distinct recovery began at 72 h post-exposure; it was progressive and complete by 14 days in the case of serum. However, the brain AChE inhibition recovery was r a t h e r slow and not complete even at 21 days, the enzyme activity being less than 70% of the control. Control values of enzyme activity at various points are given in Table II. Throughout the experimental period the rats were normal, showed no symptoms nor were their food intake or body weights affected. Subacute exposure and ChE inhibition Daily dermal application of 50 mg/kg body weight of Bromophos for 5 and 10 days, produced marked inhibition of serum pChE (> 70%) and brain AChE
203
T A B L E II S E R U M C H O L I N E S T E R A S E A N D B R A I N A C E T Y L C H O L I N E S T E R A S E A C T I V I T I E S OF CONT ROL ( V E H I C L E - T R E A T E D ) F E M A L E R A T S IN A C U T E , T I M E - C O U R S E , A N D SUBACUTE DERMAL EXPOSURE/POST-EXPOSURE PERIODS
Experiment
Serum pChE
Brain A C h E
0.954 -+ 0.022 0.864 _+ 0.068
1.535 -+ 0.092 1.321 _+ 0.056
1.156 1.242 1.018 1.118 1.044 1.017 0.994 1.012
1.309 1.535 1.558 1.609 1.603 1.378 1.377 1.431
Time course
(a) Exposure: (h) 4,
8
16, 24 (b) Post-exposure: (days) 1 2
3 6 7 10 14 21
_+ _+ -+ + -+ -
0.094 0.022 0.064 0.074 0.118 0.042 0.016 0.051
_+ _ _+ -+ -+
0.135 0.136 0.023 0.147 0.187 0.033 0.034 0.092
Subacute
(a) Exposure: (days) 5
10 (bl Post-exposure: (days) 5 10 15
0.894 _+ 0.104 1.106 +_ 0.062
1.377 _+ 0.055 1.625 _+ 0.189
1.104 _+ 0.180 0.908 -+ 0.119 0.986 -+ 0.124
1.625 _- 0.258 1.269 -+ 0.015 1.214 - 0.014
Values (~mol min -1 g-1 or m1-1) represent mean activities _+ S.E. of the mean of 4 control rats killed at each time period.
(61--70O/o) (Fig. 3). Recovery of the enzyme inhibition in both the serum and brain was rapid as seen at 5 days post-exposure, the serum pChE returning to near control values at 10 days post-exposure, whereas the brain enzyme recovery was 75% of the control at 15 days of post-exposure period (Fig. 3). In another experiment employing very low dosages of dermal exposure of Bromophos at 10, 25 and 50 mg/kg body weight for 17 days, there was doserelated inhibition of the serum pChE which ranged from 5 3 - 6 6 % (Fig. 4). The activity, 15 days post-exposure had completely recovered to the control levels. Control values of enzyme activity at various time intervals are shown in Table II. DISCUSSION
The results indicate very low acute dermal toxicity of Bromophos to the female rat, as it was not lethal up to 4000 mg/kg body weight. This also confirms the observation of Gaines et al. [10] who reported that Bromophos
204
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Days Fig. 3. Serum cholinesterase and brain acetylcholinesterase of female rats treated dermally with 50 mg/kg body weight of Bromophos for 5 and 10 days and followed by 5, 10 and 15 days of postexposure recovery period. Each value (% control) is a Mean ± S.E. (vertical bars) of 4 rats/ treatment group (See Table II for control values of enzyme activity).
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Fig. 4. Serum cholinesterase of female rats given daily dermal applications of Bromophos at 10, 25 and 50 mg/kg body weight for 17 days and after 15 days of post-exposure recovery period. Values (% of control) are Mean ± S.E. of 4 animals/treatment group.
205
was not lethal up to 5000 mg/kg body weight through the dermal route. However, appearance of symptoms beyond 2000 mg/kg body weight indicate cholinergic stimulation. The cholinesterase inhibition studies show very high sensitivity of the plasma or serum pChE to inhibition as compared to that of brain and erythrocyte AChE as seen from the in vivo ID5o values, and timecourse studies. Our results are at variance with those of Muacevic et al. [8] who reported that oral administration of Bromophos (0.5 LDs0) to rats caused maximal inhibition of the serum ChE at 8 h and complete regeneration of the enzyme in 3 days. We have found maximum inhibition of serum cholinesterase at 16 h after the dermal application of a single dose of Bromophos and complete recovery of the enzyme occurs only by 14 days post-exposure. Further, Muacevic et al. [8] reported less than 50O/o inhibition of serum ChE at 0.5 LDs0 dose of Bromophos, whereas, we got 86O/o inhibition at a dose less than 1/5 LDso. The possibility that this discrepancy could be due to different routes of exposure is remote, since our studies have shown that oral administration of acute doses of Bromophos to rats do not reveal significant differences from those of acute dermal exposure in the degree of cholinesterase inhibition in serum and brain [17]. Our studies also point out that high inhibition of AChE in the brain (> 65°/o) in acute dermal studies, coincides with the onset of symptoms which have also been observed after acute oral administration of Bromophos [17]. This is in contrast to the observation of Muacevic et al. [8] who concluded that there was no relation between the cholinesterase inhibition and symptoms or mortality. Their conclusion was based on data which showed no dose-dependent increase of AChE from 1 0 0 - 4 0 0 0 mg/kg body weight of Bromophos. This discrepancy could perhaps be attributed to the titrimetric and manometric methods of assays and consequent high variation in their results [14,18]. In our studies, dose-ChE response is clearly evident (Fig. 1). Stiasni et al. [19] using radiolabelled Bromophos have shown that Bromophos is rapidly absorbed from the gut and metabolised with an halflife of 14 h in rats. Similar studies on percutaneous uptake have not been reported for Bromophos. However, the onset of ChE inhibition in the serum as well as in brain earlier than 4 h, in our study, suggests the rapid absorption and metabolism since the parent thioate has to undergo oxidative desulfuration to produce the metabolite, Bromoxon, the potent cholinesterase inhibitor [8,9,20]. Recent studies of Knaak et al. [20] have shown that [14C]parathion, an organophosphorus insecticide, is rapidly absorbed percutaneously reaching maximum concentration in tissues and blood at 4 h which is sustained up to 12 h in the rat. It is likely that Bromophos, being a highly lipophilic organophosphorus compound, is absorbed through the skin rapidly and metabolised which is reflected in the rapid and high inhibition of ChE. In subacute studies, though high inhibition of serum pChE and brain AChE was seen, the inhibition was reversible on cessation of exposure without leaving deleterious effects. The rate of ChE recovery in rats treated with repeated doses of Bromophos was faster than in acute studies which
206
could be due to the synthesis of the new enzyme protein as well as the reactivation of the inhibited enzyme [21,22]. Absence of symptoms despite high inhibition of AChE in the brain of rats in subacute studies as in the case of other organophosphates, suggests a biochemical adaptation [21--23]. Our studies have also shown that repeated dermal exposure to very low levels of Bromophos (10 mg/kg body weight - 2 mg/rat) is reflected in marked depression of the serum pChE which is reversible on cessation of exposure. Based on these results and the demonstration of in vivo susceptibility of serum pChE in acute studies, the serum cholinesterase appears to be the most sensitive diagnostic parameter for monitoring Bromophos exposure. ACKNOWLEDGEMENTS
The authors wish to thank Mr. S.K. Majumder and Dr. B.L. Amla, for their keen interest and Dr. K. Visweswariah for kindly supplying the technical Bromophos. We appreciate the help-of Mr. K. Nanjundiah in the preparation of figures and photographs, Mr. B.S. Ramesh for statistical analyses and Mr. Viswanatha for his assistance in autopsy and haematology. The second author (PJ) was the recipient of CSIR (India) Junior research fellowship. REFERENCES 1 2 3 4 5 6 7
8 9 10 ll 12 13 14
R. Immel and G. Geisthardt, Bromophos, ein neus Insektizid mit geringer Toxizitiit. Mededel. Landbouwhogeschool Opzoekingsta., Staat Gent, 29 (1964) 1242. R.O. Drummond, Further evaluation of animal systemic insecticides., J. Econ. Entomol., 60 (1966) 733. D. Eichler, Bromophos and bromophos-ethyl residues. Res. Rev., 41 (1972) 65. W.H.0. 1972. Evaluations of some pesticide residues in food. W.H.O. Pesticide Residues Series No. 2 (1973) 7. J. Kinkel, G. Muacevic, R. Sehring and G. Bodenstein, Zur Toxicologie yon Bromophos (O,ODimethyl O-(2,5-dichlor-4-bromophenyl)-thionophosphat). Arch. Toxicol., 22 (1966) 36. E. D6hring, Mittel gegen Gesundheits und Haussch~id linge. Zur Toxicologie und Anwendung. B. Gesundh. BI. 10 (1967) 129 und 150. K. Liesche, Insectizide, akarizide und fungizide Ester und Amide der Phosphors~iuren, Polyphosphors~uren, substituierten Phosphorsiiuren und anderer Phosphorsiiuren. Dtsch. Apotheker Ztg., 106 (1966) 350. G. Muacevic, E. Dirks, H.J. Kinkel and F. Leuschner, Anticholinesterase activity of Bromophos in rats and dogs. Toxicol. Appl. Pharmacol., 16 (1970) 585. J. Stenersen, Dimethylation of the insecticide Bromophos by a glutathione-dependent liver enzyme and by alkaline buffers. J. Econ. Entomol., 62 (1969) 1043. T.B. Gaines, Acute toxicity of pesticides. Toxicol. Appl. Pharmacol., 14 (1969) 515. S. Rajendran and M. Muthu, Fumigant action of Bromophos tested against Tribolium castaneum, Central Food Tech. Res. Institute (Mysore) Annu. Rep. (1985--86). A. Pasha, K. Visweswariah and S.K. Majumder. Synthesis and bench-scale production of Bromophos. Central Food Tech. Res. Inst., (Mysore), Annu. Rep. (1984-85). G.L. Ellman, D.K. Courtney, V. Andres and R.M. Featherstone, A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol., 7 (1961) 88. A. Silver, The Biology of Cholinesterases, Elsevier, Amsterdam, 1974, p. 63.
207
15 16 17 18 19
20
21 22 23
208
G.W. Snedecor and W.G. Cochran, Statistical Methods, Iowa State University Press, Ames, Iowa, 1967, p. 91. D.J. Finney, Probit Analysis, Cambridge University Press, Cambridge, 1977, p. 50. T. Shivanandappa, Acute oral toxicity and anticholinesterase effects of Bromophos in the albino rat. Central Food Toch. Res. Inst. (Mysorel, Ann. Rep. (1985--86~. K.B. Augustinsson, Determination of cholinesterases in D. Glick (Ed.), Methods of Biochemical Analysis. Suppl. volume, Interscienee, New York, 1971, p. 217. Stiasni, D. Rehbinder and W. Deckers, Absorption, distribution and metabolism of 0-(4bromo-2,5-dichlorophenyl)-O,O-dimethylphosphorothioate (Bromophos)in the rat. J. Agric. Food Chem., 15 (1967~ 474. J.B. Knaak, K. Yee, C.R. Ackerman, G. Zweig, D.M. Fry and B.W. Wilson. Percutaneous absorption and dermal dose-Cholinesterase response studies with parathion and carbaryl in the rat. Toxicol. Appl. Pharmacol., 76 (1984) 252. D.F. Heath, Organophosphorus Poisons, Pergamon Press, Oxford, 1961, p. 177. D.J. Ecobichon, A.M. Comeau and P.H. Cameron, Chronic toxicity of technical Fenitrothion in male rats. Toxicol. Appl. Pharmacol., 56 (1980) 409. B. Trottier, A.R., Fraser, G. Planet and D.J. Eeobichon, Subacute toxicity of technical Fenitrothion in male rats. Toxicology, 17 (1980) 29.
R E S P O N S E O F B L O O D A N D B R A I N C H O L I N E S T E R A S E TO D E R M A L E X P O S U R E O F B R O M O P H O S IN T H E R A T
T. SHIVANANDAPPA*,PIUS JOSEPH and M.K. KRISHNAKUMARI Toxicology Uni~ Infestation Control and Protectants Discipline, Central Food Technological Research Institute, Mysore - 570 013 (India)
(Received April 2nd, 1987} (Accepted September 13th, 1987) SUMMARY
Response of cholinesterase to dermal exposure of acute, single and multiple doses of Bromophos in the female rat has been studied. Doseresponse studies (50--4000 mg/kg body weight, 24 h exposure) showed that plasma cholinesterase was most sensitive to inhibition in vivo, followed by the brain and erythrocyte acetylcholinesterase. The ID5o values for the in vivo cholinesterase inhibition were 10.1, 576.1 and 1938.0 mg/kg body weight for the plasma, brain and erythrocytes, respectively. In time-course studies after a single sublethal dose of 1000 mg/kg body weight (24 h) of Bromophos, the serum and brain cholinesterase were rapidly inhibited reaching a maximum at 16 h. Recovery was complete in the case of serum at 14 days post-exposure, whereas the brain enzyme was not fully recovered at 21 days. In a subacute study, daily dermal application of 50 mg/kg body weight of Bromophos for 5 and 10 days, resulted in high inhibition of the serum cholinesterase and brain acetylcholinesterase, the former being more marked which was reversible after 10 or 15 days of post-exposure period. Very low levels of dermal exposure of Bromophos (10--50 mg/kg body weight) for 17 days caused pronounced depression of serum cholinesterase which completely recovered in 15 days after cessation of exposure suggesting that the serum cholinesterase could serve as the most sensitive diagnostic indicator of Bromophos exposure. w o r d s : Bromophos; O,O-Dimethyl-O(2,5-dichloro-4-bromophenyl}-phosphorothioate; Pseudocholinesterase; Acetylcholinesterase; Brain; Erythrocytes; Dermal toxicity
Key
*To whom all correspondence and reprint requests should be addressed. ACHE, acetylcholinesterase; Bromophos, [O,O-dimethyl-O-(2,5-dichloro-4bromophenyt) phosphorothioate]; pChE, pseudocholinesterase.
Abbreviations:
0300-483X/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
199
INTRODUCTION
Bromophos [O, O-dimethyl-O-(2,5-dichloro-4-bromophenyl)phosphorothioate] is a non-systemic organophosphorus insecticide widely used in crop protection, infestation control of stored grain, public health and on ticks and mites of domestic animals [1--4]. One distinctive feature of Bromophos, among organophosphorus insecticides, is its extremely low acute toxicity to mammals with an LD~0 of 3700--6100 mg/kg body weight in the rat and 3311 --6000 mg/kg body weight in mice [5--7]. It is quickly absorbed and excreted when ingested orally with a biological half-life of 14 h in the rat [20]. Bromophos undergoes metabolic activation and is an inhibitor of acetylcholinesterase in vivo in mammals [8,9]. Because of its very low acute toxicity, Bromophos has been specially recommended for the ectoparasite control of domestic animals [3]. Gaines [10] has reported that acute dermal LDs0 for Bromophos lies beyond 5000 mg/kg body weight to rats but there is no information on the response of cholinesterase to dermal exposure. Since Bromophos is a solid at room temperature with a negligible vapour pressure [11], its main route to occupational exposure to handlers and agricultural workers will be through percutaneous absorption. Hence, for an exposure risk assessment and monitoring, it is essential to understand the effects of Bromophos after dermal application. The present study was therefore undertaken to investigate the acute and subacute toxicity of Bromophos with reference to cholinesterase inhibition and recovery in the blood and brain. MATERIALS AND METHODS
A n i m a l s and t r e a t m e n t
Female adult albino rats of Wistar strain weighing 1 8 0 - 2 0 0 g were housed in individual cages, grouped into 4 rats/group and provided with food and water ad lib. The skin on the dorsal side behind the neck was shaved with an electrically operated hair clipper. The clipped area was 3 × 2 cm. Technical bromophos (93% pure) was synthesised in this Institute which was identical to the authentic Bromophos {Environmental Protection Agency, U.S.A.; and CelaMerck, Darmstadt, Germany) in terms of physical, chemical and insecticidal properties [12]. Bromophos {solid, white crystalline powder) was dissolved in ethyl acetate and applied evenly as a thin layer on the shaven skin surface. Control rats were treated with ethyl acetate alone. For acute dose-response studies, doses of Bromophos ranging from 50 to 4000 mg/kg body weight in a maximum of 0.5 ml of the carrier were applied and after 24 h of exposure, the animals were sacrificed. For time-course studies, a single acute sublethal dose of 1000 mg/kg body weight was applied, rats were killed at various time intervals of 4, 8, 16 and 24 h. For recovery studies groups of rats after 24 h of dermal exposure to Bromophos, the applied area of the skin was cleaned with alcohol and soapy water and the animals killed at 1, 2, 3, 6, 7, 10, 14 and 21 days. Two subacute studies were made: (i) daily dermal application of 50
200
mg/kg body weight of Bromophos in 0.2 ml of ethyl acetate for 5 and 10 days with a recovery period of 5, 10 and 15 days after the 10th application; and (ii) daily application of 10, 25 and 50 mg/kg body weight of Bromophos for 17 days and a recovery period of 15 days after the last application to monitor the serum cholinesterase as a diagnostic indicator of low level of exposure to Bromophos.
Sample preparation Sacrifice of animals was done after diethyl ether anaesthesia. For the separation of erythrocytes, blood was drawn in heparinised syringes, centrifuged at 1000 x g. and the plasma was collected; the sediment was washed twice with 0.9% saline and the erythrocytes were suspended in saline to the original blood volume. At the time of assay for acetylcholinesterase, an aliquot of the erythrocyte suspension was lysed with distilled water and used as the enzyme source. To obtain serum, blood was collected directly from the heart without any anticoagulant, allowed to clot and then centrifuged at 1000 x g. for 15 rain. Brains were removed, washed in saline and homogenised in 0.1 M ice-cold phosphate buffer (pH 7.4) using a Potter-Elvehjem motor-driven homogeniser, centrifuged at 1000 x g. and the supernatant was used for acetylcholinesterase assays.
Enzyme assays Pseudocholinesterase (EC 3.1.1.8 pChE) in the plasma or serum, and acetylcholinesterase (EC 3.1.1.7 ACHE) in the brain and erythrocytes were assayed by the method of Ellman et al. [13] with acetylthiocholine iodide (Merck, Darmstadt, F.R.G.) as the substrate by recording the change in absorbance every 30 s for 3 rain at 412 nm. The enzyme activity was calculated by the extinction coefficient (~:412 ~-~ 1.36 x 10~ M -1 cm-9 for the yellow anion, 5,dithio-2, nitrobenzoid acid [14], and expressed as pmol min -~ or percent of control.
Statistical analyses Differences between the treated and control groups were analysed by Student's' t-test [15]. Significance was assessed at 0.1 or 1% level (P < 0.001 or < 0.01L In vivo IDs0 were calculated from the dose-response data of cholinesterase inhibition by probit regression analysis [16]. RESULTS
Dose-response study There was no mortality up to the highest dosage (4000 mg/kg body weight) of Bromophos. However, mild symptoms appeared at 2000 mg/kg body weight and were well pronounced at 4000 mg/kg body weight. The symptoms were typically cholinergic such as lacrimation, salivation, urination and nasal discharge which were mild at 8 h of exposure, distinct at 24 h, lasted up to 48 h and by 72 h, the rats had completely recovered. Except in the highest dose, food intake was not affected.
201
• Plasma [ ] Brain [ ] Erythrocyte
100
8
~60 ..j 1::3
LtJ
4O
e-
t
20
50
100
250
500
1000
2000
4000
Dose (mg/kg b.w.) Fig. 1. Dose-response of plasma cholinesterase, erythrocyte and brain acetylcholinesterase inhibition in female rats after a 24 h acute dermal exposure to Bromophos. Each value (% of control) is the Mean _+ S.E. (vertical bars) of 4 animals/group.
pChE in the plasma and, AChE in erythrocytes and brain showed dosedependent inhibition (Fig. 1). Marked inhibition (61o/o) of pChE was seen even at the lowest dosage and it was maximum (86O/o) at 2000 and 4000 mg/kg body weight. No significant inhibition of AChE in the brain was seen at the lowest dosage, but it was dose-dependent beyond 100 mg/kg body weight which ranged from 360/o to 67o/o (Fig. 1). It may be noted that inhibition did not increase beyond 2000 mg/kg body weight and the onset of symptoms was evident with the high AChE inhibition in the brain. Erythrocyte AChE was not affected at 50 and 100 mg/kg body weight but was inhibited beyond 250 TABLE I IN VIVO SENSITIVITY OF CHOLINESTERASE TO ACUTE DERMAL BROMOPHOS IN THE FEMALE RAT Enzyme source
IDa0*
Probit regression equation**
Plasma Brain Erythrocyte
10.1 567.1 1938.0
Y = 4.5155 + 0.4811 x Y = 2.0718 + 1.0634 x Y = 1.7792 + 0.0798 x
DOSES
OF
*The dose (mg/kg body weight for 24 h) required to produce 50% inhibition of cholinesterase in vivo. **Values were computed by probit regression analysis from the data shown in Fig. 1.
202
~-
100
Brain
4- S e r u m
-~ 80 0 c.3
"S 60
>
40
Ld
~- 2 0
{_)
"[
1
8 1'6 24 2 3 ~ 7 110 1'4 21 - -
h rs - -
-
-
,days
Fig. 2. Time course of inhibition of serum cholinesterase (pChE) and brain acetyl cholinesterase (ACHE) in female rats after a single acute dermal application of a sublethal dose (1000 mg/kg body weight) of Bromophos. Values (% of control) at each time interval are expressed as Mean +_ S.E. (vertical bars) of 4 animals. (See Methods for experimental details and Table II for control values of cholinesterase).
mg/kg body weight and the inhibition progressed beyond 2000 mg/kg body weight unlike the plasma and brain enzyme (Fig. 1). IDs0 values show vast differences of the cholinesterase of the plasma, brain, and e r y t h r o c y t e s in their susceptibility to inhibition in vivo by Bromophos, viz., 10.1, 567.1 and 1938 mg/kg body weight, respectively (Table I).
Time-course of cholinesterase inhibition and recovery Figure 2 depicts the time-course inhibition and recovery pattern of serum pChE and brain AChE after 24 h dermal exposure to a single sublethal dose (1000 mg/kg body weight) of Bromophos. The inhibition in both serum and brain could be seen as early as 4 h which progressed to reach a maximum at 16 h of exposure and it was unchanged up to 24 h. Distinct recovery began at 72 h post-exposure; it was progressive and complete by 14 days in the case of serum. However, the brain AChE inhibition recovery was r a t h e r slow and not complete even at 21 days, the enzyme activity being less than 70% of the control. Control values of enzyme activity at various points are given in Table II. Throughout the experimental period the rats were normal, showed no symptoms nor were their food intake or body weights affected. Subacute exposure and ChE inhibition Daily dermal application of 50 mg/kg body weight of Bromophos for 5 and 10 days, produced marked inhibition of serum pChE (> 70%) and brain AChE
203
T A B L E II S E R U M C H O L I N E S T E R A S E A N D B R A I N A C E T Y L C H O L I N E S T E R A S E A C T I V I T I E S OF CONT ROL ( V E H I C L E - T R E A T E D ) F E M A L E R A T S IN A C U T E , T I M E - C O U R S E , A N D SUBACUTE DERMAL EXPOSURE/POST-EXPOSURE PERIODS
Experiment
Serum pChE
Brain A C h E
0.954 -+ 0.022 0.864 _+ 0.068
1.535 -+ 0.092 1.321 _+ 0.056
1.156 1.242 1.018 1.118 1.044 1.017 0.994 1.012
1.309 1.535 1.558 1.609 1.603 1.378 1.377 1.431
Time course
(a) Exposure: (h) 4,
8
16, 24 (b) Post-exposure: (days) 1 2
3 6 7 10 14 21
_+ _+ -+ + -+ -
0.094 0.022 0.064 0.074 0.118 0.042 0.016 0.051
_+ _ _+ -+ -+
0.135 0.136 0.023 0.147 0.187 0.033 0.034 0.092
Subacute
(a) Exposure: (days) 5
10 (bl Post-exposure: (days) 5 10 15
0.894 _+ 0.104 1.106 +_ 0.062
1.377 _+ 0.055 1.625 _+ 0.189
1.104 _+ 0.180 0.908 -+ 0.119 0.986 -+ 0.124
1.625 _- 0.258 1.269 -+ 0.015 1.214 - 0.014
Values (~mol min -1 g-1 or m1-1) represent mean activities _+ S.E. of the mean of 4 control rats killed at each time period.
(61--70O/o) (Fig. 3). Recovery of the enzyme inhibition in both the serum and brain was rapid as seen at 5 days post-exposure, the serum pChE returning to near control values at 10 days post-exposure, whereas the brain enzyme recovery was 75% of the control at 15 days of post-exposure period (Fig. 3). In another experiment employing very low dosages of dermal exposure of Bromophos at 10, 25 and 50 mg/kg body weight for 17 days, there was doserelated inhibition of the serum pChE which ranged from 5 3 - 6 6 % (Fig. 4). The activity, 15 days post-exposure had completely recovered to the control levels. Control values of enzyme activity at various time intervals are shown in Table II. DISCUSSION
The results indicate very low acute dermal toxicity of Bromophos to the female rat, as it was not lethal up to 4000 mg/kg body weight. This also confirms the observation of Gaines et al. [10] who reported that Bromophos
204
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0
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i~
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60
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,I,',J
/ih
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I
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.
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,
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,
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,'/11
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V:V, -II
1'1
' ,. I! Ii
10+10
10+15
Days Fig. 3. Serum cholinesterase and brain acetylcholinesterase of female rats treated dermally with 50 mg/kg body weight of Bromophos for 5 and 10 days and followed by 5, 10 and 15 days of postexposure recovery period. Each value (% control) is a Mean ± S.E. (vertical bars) of 4 rats/ treatment group (See Table II for control values of enzyme activity).
i lO0
I--I I O m g l k g b.w. r"?;I 25 mg I kg b.w. 5 0 m g l k g b.w.
80
i I I i :p-,;, ~ill - :.' ': c' /lll~:;:,,.J
0
6O
, ii.
°--
t L~ t'-
~
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,/i, ,s.....,, I , I i " i ~;'.,~
0
(.)
,.//
" i l il;./?
:.,.i;il ;,:l
I I II1,:,~i'. t
',;(,I?-Y "Ii/L":'::'I~1 'Y,'/I: :.-:1 Exposure
i/// ~' ,, il II ',,'" i / i / ,"," l / 1 / ' , ? i' ( I/7/
//Ii ,/it/ V/l~ IIIII
' "~-~",
":,'~ !"(7~1 ~., ,.,' ,< ,+, ::_.:
Recovery
Fig. 4. Serum cholinesterase of female rats given daily dermal applications of Bromophos at 10, 25 and 50 mg/kg body weight for 17 days and after 15 days of post-exposure recovery period. Values (% of control) are Mean ± S.E. of 4 animals/treatment group.
205
was not lethal up to 5000 mg/kg body weight through the dermal route. However, appearance of symptoms beyond 2000 mg/kg body weight indicate cholinergic stimulation. The cholinesterase inhibition studies show very high sensitivity of the plasma or serum pChE to inhibition as compared to that of brain and erythrocyte AChE as seen from the in vivo ID5o values, and timecourse studies. Our results are at variance with those of Muacevic et al. [8] who reported that oral administration of Bromophos (0.5 LDs0) to rats caused maximal inhibition of the serum ChE at 8 h and complete regeneration of the enzyme in 3 days. We have found maximum inhibition of serum cholinesterase at 16 h after the dermal application of a single dose of Bromophos and complete recovery of the enzyme occurs only by 14 days post-exposure. Further, Muacevic et al. [8] reported less than 50O/o inhibition of serum ChE at 0.5 LDs0 dose of Bromophos, whereas, we got 86O/o inhibition at a dose less than 1/5 LDso. The possibility that this discrepancy could be due to different routes of exposure is remote, since our studies have shown that oral administration of acute doses of Bromophos to rats do not reveal significant differences from those of acute dermal exposure in the degree of cholinesterase inhibition in serum and brain [17]. Our studies also point out that high inhibition of AChE in the brain (> 65°/o) in acute dermal studies, coincides with the onset of symptoms which have also been observed after acute oral administration of Bromophos [17]. This is in contrast to the observation of Muacevic et al. [8] who concluded that there was no relation between the cholinesterase inhibition and symptoms or mortality. Their conclusion was based on data which showed no dose-dependent increase of AChE from 1 0 0 - 4 0 0 0 mg/kg body weight of Bromophos. This discrepancy could perhaps be attributed to the titrimetric and manometric methods of assays and consequent high variation in their results [14,18]. In our studies, dose-ChE response is clearly evident (Fig. 1). Stiasni et al. [19] using radiolabelled Bromophos have shown that Bromophos is rapidly absorbed from the gut and metabolised with an halflife of 14 h in rats. Similar studies on percutaneous uptake have not been reported for Bromophos. However, the onset of ChE inhibition in the serum as well as in brain earlier than 4 h, in our study, suggests the rapid absorption and metabolism since the parent thioate has to undergo oxidative desulfuration to produce the metabolite, Bromoxon, the potent cholinesterase inhibitor [8,9,20]. Recent studies of Knaak et al. [20] have shown that [14C]parathion, an organophosphorus insecticide, is rapidly absorbed percutaneously reaching maximum concentration in tissues and blood at 4 h which is sustained up to 12 h in the rat. It is likely that Bromophos, being a highly lipophilic organophosphorus compound, is absorbed through the skin rapidly and metabolised which is reflected in the rapid and high inhibition of ChE. In subacute studies, though high inhibition of serum pChE and brain AChE was seen, the inhibition was reversible on cessation of exposure without leaving deleterious effects. The rate of ChE recovery in rats treated with repeated doses of Bromophos was faster than in acute studies which
206
could be due to the synthesis of the new enzyme protein as well as the reactivation of the inhibited enzyme [21,22]. Absence of symptoms despite high inhibition of AChE in the brain of rats in subacute studies as in the case of other organophosphates, suggests a biochemical adaptation [21--23]. Our studies have also shown that repeated dermal exposure to very low levels of Bromophos (10 mg/kg body weight - 2 mg/rat) is reflected in marked depression of the serum pChE which is reversible on cessation of exposure. Based on these results and the demonstration of in vivo susceptibility of serum pChE in acute studies, the serum cholinesterase appears to be the most sensitive diagnostic parameter for monitoring Bromophos exposure. ACKNOWLEDGEMENTS
The authors wish to thank Mr. S.K. Majumder and Dr. B.L. Amla, for their keen interest and Dr. K. Visweswariah for kindly supplying the technical Bromophos. We appreciate the help-of Mr. K. Nanjundiah in the preparation of figures and photographs, Mr. B.S. Ramesh for statistical analyses and Mr. Viswanatha for his assistance in autopsy and haematology. The second author (PJ) was the recipient of CSIR (India) Junior research fellowship. REFERENCES 1 2 3 4 5 6 7
8 9 10 ll 12 13 14
R. Immel and G. Geisthardt, Bromophos, ein neus Insektizid mit geringer Toxizitiit. Mededel. Landbouwhogeschool Opzoekingsta., Staat Gent, 29 (1964) 1242. R.O. Drummond, Further evaluation of animal systemic insecticides., J. Econ. Entomol., 60 (1966) 733. D. Eichler, Bromophos and bromophos-ethyl residues. Res. Rev., 41 (1972) 65. W.H.0. 1972. Evaluations of some pesticide residues in food. W.H.O. Pesticide Residues Series No. 2 (1973) 7. J. Kinkel, G. Muacevic, R. Sehring and G. Bodenstein, Zur Toxicologie yon Bromophos (O,ODimethyl O-(2,5-dichlor-4-bromophenyl)-thionophosphat). Arch. Toxicol., 22 (1966) 36. E. D6hring, Mittel gegen Gesundheits und Haussch~id linge. Zur Toxicologie und Anwendung. B. Gesundh. BI. 10 (1967) 129 und 150. K. Liesche, Insectizide, akarizide und fungizide Ester und Amide der Phosphors~iuren, Polyphosphors~uren, substituierten Phosphorsiiuren und anderer Phosphorsiiuren. Dtsch. Apotheker Ztg., 106 (1966) 350. G. Muacevic, E. Dirks, H.J. Kinkel and F. Leuschner, Anticholinesterase activity of Bromophos in rats and dogs. Toxicol. Appl. Pharmacol., 16 (1970) 585. J. Stenersen, Dimethylation of the insecticide Bromophos by a glutathione-dependent liver enzyme and by alkaline buffers. J. Econ. Entomol., 62 (1969) 1043. T.B. Gaines, Acute toxicity of pesticides. Toxicol. Appl. Pharmacol., 14 (1969) 515. S. Rajendran and M. Muthu, Fumigant action of Bromophos tested against Tribolium castaneum, Central Food Tech. Res. Institute (Mysore) Annu. Rep. (1985--86). A. Pasha, K. Visweswariah and S.K. Majumder. Synthesis and bench-scale production of Bromophos. Central Food Tech. Res. Inst., (Mysore), Annu. Rep. (1984-85). G.L. Ellman, D.K. Courtney, V. Andres and R.M. Featherstone, A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol., 7 (1961) 88. A. Silver, The Biology of Cholinesterases, Elsevier, Amsterdam, 1974, p. 63.
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15 16 17 18 19
20
21 22 23
208
G.W. Snedecor and W.G. Cochran, Statistical Methods, Iowa State University Press, Ames, Iowa, 1967, p. 91. D.J. Finney, Probit Analysis, Cambridge University Press, Cambridge, 1977, p. 50. T. Shivanandappa, Acute oral toxicity and anticholinesterase effects of Bromophos in the albino rat. Central Food Toch. Res. Inst. (Mysorel, Ann. Rep. (1985--86~. K.B. Augustinsson, Determination of cholinesterases in D. Glick (Ed.), Methods of Biochemical Analysis. Suppl. volume, Interscienee, New York, 1971, p. 217. Stiasni, D. Rehbinder and W. Deckers, Absorption, distribution and metabolism of 0-(4bromo-2,5-dichlorophenyl)-O,O-dimethylphosphorothioate (Bromophos)in the rat. J. Agric. Food Chem., 15 (1967~ 474. J.B. Knaak, K. Yee, C.R. Ackerman, G. Zweig, D.M. Fry and B.W. Wilson. Percutaneous absorption and dermal dose-Cholinesterase response studies with parathion and carbaryl in the rat. Toxicol. Appl. Pharmacol., 76 (1984) 252. D.F. Heath, Organophosphorus Poisons, Pergamon Press, Oxford, 1961, p. 177. D.J. Ecobichon, A.M. Comeau and P.H. Cameron, Chronic toxicity of technical Fenitrothion in male rats. Toxicol. Appl. Pharmacol., 56 (1980) 409. B. Trottier, A.R., Fraser, G. Planet and D.J. Eeobichon, Subacute toxicity of technical Fenitrothion in male rats. Toxicology, 17 (1980) 29.