Cholinesterase Activities in the Blood and Brain of Rats and Mice, as Determined by a Rapid

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1 industrial Health, 1985, 23, ORIGINAL ARTICLES Cholinesterase Activities in the Blood and Brain of Rats and Mice, as Determined by a Rapid Colorimetric Method Katsumaro TOMOKUNI and Tohru HASEGAWA Department of Community Health Science, Saga Medical School, Nabeshima, Saga , Japan (Received November 19, 1984) Abstract : The erythrocyte, plasma and brain cholinesterase (ChE) activities of male rats and mice were determined using a simple colorimetric micromethod. In the normal male mice, the plasma ChE activity was 2.3 times higher than that of the erythrocyte. On the contrary, in the normal male rats, the erythrocyte ChE activity was 2.3 times higher than that of the plasma. The brain ChE activity was similar between rats and mice. The erythrocyte, plasma and brain ChE activities were decreased in both rats and mice exposed to diazinon and, however, this enzyme inhibition was most remarkable in the plasma of mice. Key words: ChE activity-blood and brain Rapid measurement-diazinon toxicit INTRODUCTION The determination of blood cholinesterase (ChE) activity plays an important role for the biological monitoring of health effects of organophosphate and carbamate insecticides in men and animals. Up to date, a great variety of methods has been described on the measurement of erythrocyte and plasma ChE activities. An electrometric method was reported by Miche1.1) A radioisotope method using acetylcholine-14c was introduced by Winteringham and Disney.2) However, the above methods are unsuitable for routine measurement of ChE activity because they require a lot of time. In 1961, Ellman et al.3) have demonstrated a new colorimetric method for determining ChE activity using acetylthiocholine iodide and dithiobisnitrobenzoic acid. This new method is rapid and extremely sensitive, and further it is applicable to small amounts of sample. After that, this method has been modified by several investigators.4-6)

2 76 K. TOMOKUNI AND T. HASEGAWA In present paper, we tried to modify the colorimetric methods described above for more simple determination of ChE activity in the blood and brain of laboratory animals. This modified method was applied to ChE assay in normal diazinonexposed animals and the availability of our method was confirmed. MATERIALS AND METHODS Animals and Sample Collection: Male Wistar rats ( g body wt.) and male DDY mice (34-38 g body wt.) were used as the experimental animals. Animals were divided into two groups. One group was used as the control. Another group received a single intraperitoneal injection of diazinon (97% pure) at a dose of 20 or 100 mg/kg body wt. The animal was light anesthetized with ethyl ether and the blood was withdrawn into a glass test-tube through a heparinized capillary tube by the orbital bleeding technique. After decapitation, the brain was excised and rinsed well in an ice-cold 0.9% sodium chloride solution. The samples were quickly used for the enzyme assay. Determinations of Blood and Brain Cholinesterase Activities Reagents: Distilled water and chemical reagents of analytical grade were used. Solution A-1; Dissolve 10 mg of 5,5-dithiobis-2-nitrobenzoic acid (DTNB, Wako Chemicals) in 50 ml of 0.9% sodium chloride solution and add 50 ml of 1/15 N Sorensen phosphate buffer (ph 8). Solution A-2; Dissolve 10 mg of DTNB in 100 ml of 1/15 M SOrensen phosphate buffer (ph 8). These solutions were stable more than one week at 5 Ž. Solution B; Dissolve 75 mg of acetylthiocholine iodide (Sigma Chemical Co.) in 50 ml of water. This substrate solution must always be freshly prepared and should be kept in an ice bath during a day's work. Solution C; Dissolve 50 mg of eserine salicylate (Sigma Chemical Co.) in 50 ml of water. Place this solution into a dropping bottle and store in a refrigerator. Procedure. 1) Blood ChE activity was determined according to the procedure presented in Fig. 1. In this procedure, the absorbance of the yellow color in the tube No. 1 is a measure of total ChE activity in the whole blood. The absorbance in the tube No. 2 corresponds to a measure of plasma ChE activity. On the other hand, erythrocyte ChE activity is determined by subtracting the plasma ChE activity value from that of total ChE. The activity was expressed as micromoles of thiocholine produced per min per ml of whole blood and it was calculated according to the following formula: The value of 1.36 ~ 104 is the extinction coefficient of nitrothiobenzoate ion

3 CHOLINESTERASE ACTIVITIES OF BLOOD AND BRAIN 77 Fig. 1. Procedure for the measurement of cholinesterase activity in the erythrocyte and plasma. formed by the enzyme assay and the value was proposed by Ellman et al.3) The amount of nitrothiobenzoate ion is equivalent to the thiocholine produced. 2) Brain ChE activity was determined according to the procedure indicated in Fig. 2. The absorbance corresponding to the brain ChE activity is obtained by subtracting the absorbance of blank tube from that of sample tube. The activity was expressed as micromoles of thiocholine produced per min per g of tissue (wet wt.) and it was calculated according to the following formula: RESULTS AND DISCUSSION The method for determination of ChE activity presented in this paper is based on coupling of the following reactions:

4 78 K. TOMOKUNI AND T. HASEGAWA Fig. 2. Procedure for the measurement of cholinesterase activity in the brain. enzyme thiocholine +acetate thiocholine+dithiobisnitrobenzoate-nitrothiobenzoate (yellow color) The intensity of the yellow color formed through the production of thiocholine was proportional to the time of incubation. Using the method in Fig. 1, the blood ChE activity was measured 10 times with the same blood sample taken from a male rat. The coefficient of variation (C.V.) obtained was less than 5%. Therefore, it was indicated that the present method has a satisfactory precision. The determination of normal ChE activities in the erythrocyte, plasma and brain of male rats and mice was performed using the present method. The normal values obtained are summarized in Table 1. The normal value of erythrocyte ChE activity was almost the same between male rats and male mice. On the other hand, the plasma ChE activity was extremely higher in the mice than in the rats (P < 0.001). That is, the species difference was observed in the plasma ChE activity. The ratio of erythrocyte ChE activity to plasma ChE activity was 2.3 for the male rats and 0.4 for the male mice. The brain ChE activity of rats was slightly low compared to that of mice. The erythrocyte, plasma and brain ChE activities in both rats and mice exposed to diazinon are shown in Table 2. In the mice receiving a single dose of ion

5 CHOLINESTERASE ACTIVITIES OF BLOOD AND BRAIN 79 Table 1. Comparison of cholinesterase activity in erythrocyte, plasma and brain of normal male rats and mice Table 2. Inhibition of blood and brain cholinesterase activities in male rats and mice which received a single intraperitoneal injection of diazinon in olive oil 20 mg/kg, the inhibition of ChE activity was more remarkable in the plasma than in the erythrocyte and brain. These data indicate that plasma ChE is most sensitive to diazinon in the mice. On the other hand, in the rats of 100 mg/kg dose, the degree of ChE inhibition was similar among erythrocyte, plasma and brain. In conclusion, it was estimated that the present method is available for biological monitoring of laboratory animals and human subjects exposed to organophosphate insecticides.

6 80 K. TOMOKUNI AND T. HASEGAWA ACKNOWLEDGEMENT The authors are grateful to Y. Noda, K. Ichimaru and Y. Kabashima, Students of Saga Medical School, for providing technical assistance. REFERENCES 1) Michel, H. O. (1949). An electrometric method for the determination of red blood cell and plasma cholinesterase activity, J. Lab. Clin. Med., 34, ) Winteringham, F. P. W. and Disney, R. W. (1964). A simple method for estimating blood cholinesterase activity, Lab. Practice, 13, ) Ellman, G. L., Courtney, K. D., Andres, V. and Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol., 7, 88. 4) Voss, G. and Sachsse, K. (1970). Red cell and plasma cholinesterase activities in microsamples of human and animal blood determined simultaneously by a modified acetylthiocholine/dtnb procedure, Toxicol. Appl. Pharmacol 16, ) Hill, E. F. (1979). Cholinesterase activity in Japanese quail dusted with carbaryl, Lab. Anim. Sci., 29, ) Mount, M. E., Dayton, A. D. and Oehme, F. W. (1981). Carbaryl residues in tissues and cholinesterase activities in brain and blood of rats receiving carbaryl, Toxicol. Appl. Pharmacol., 58, 282.