Effects of Chronic Poisoning by an Organophosphorus Cholinesterase

6 Effects of Chronic Poisoning by an Organophosphorus Cholinesterase

Downloaded by NORTH CAROLINA STATE UNIV on May 4, 2015 | http://pubs.acs.org Publication Date: June 1, 1966 | doi: 10.1021/ba-1966-0060.ch006

Inhibitor on Acetylcholine and Norepinephrine Content of the Brain W . B. S T A V I N O H A , J. A . RIEGER, Jr., L . C. RYAN, and P. W . S M I T H Civil Aeromedical Institute, Federal Aviation Agency, Oklahoma City, Okla.

In animals receiving daily doses of an organophosphorus cholinesterase inhibitor for 24 days, symptoms of poisoning became maximal at about the third day, declined thereafter, and became mild after 8 to 10 days. Acetylcholine content of brain tissue rose to a high level at the third day, had returned to the control level at the tenth day, and remained at this level thereafter, although brain cholinesterase activity was only 20% of the normal value from the tenth through the 24th day. It is concluded that compensatory changes in the acetylcholine content of brain tissue occur during symptomatic adaptation to low cholinesterase activity. No significant changes in brain norepinephrine content were observed.

' T h e experiments described constitute one portion of an investigation of the mechanism by which animals become adapted to the persistent depression of cholinesterase activity which can be produced by organophosphorus pesticides. The more prominent signs and symptoms of acute organophosphorus poisoning are generally well known. They consist of a complex of the actions of acetylcholine, which persists at the sites of its release when the rate of its destruction is reduced. Thus, there are effects which can be attributed to the actions of acetylcholine at autonomic ganglia, at cholinergic postganglionic autonomic nerve endings, at somatic motor nerve terminations, and within the central nervous system i n the case of 79 In Organic Pesticides in the Environment; Rosen, A., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1966.


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those inhibitors which can traverse the blood-brain barrier. Although it is possible that the commercially useful organophosphorus pesticides affect other enzyme systems or have other intrinsic pharmacological actions, these have not been adequately explored. The order of appearance and the eventual pattern and severity of the signs and symptoms depend upon the specific organophosphorus compound involved, the route of entry into the body, the size of a single dose, or the size, number, and spacing of multiple doses. In 1952, Rider, Ellinwood, and Coon (7) demonstrated that rats can become tolerant to octamethyl pyrophosphoramide ( O M P A ) . Since then it has been shown that rats can acquire tolerance to a variety of organo phosphorus cholinesterase inhibitors. Typical of such reports is that of Bombinski and DuBois (2), who administered Ο,Ο-diethyl S-2-ethyl-2mercaptoethyl phosphorodithioate (Di-Syston) to rats each day for pe riods as long as 60 days. Signs of poisoning appeared after 2 days, but began to subside after 7-10 days, even though the activity of brain cholinesterase remained at or about 2 0 % of its normal value from the 5th day onward. It is now apparent from evidence which has been accumulating since the 1950s when large-scale use of cholinesterase inhibitors as pest-control agents began, that man can become tolerant to most, if not all, of the commercially important organophosphorus pesticides. It is not unusual for a person who has become acutely i l l from a single large dose or a series of smaller doses of a persistent cholinesterase inhibitor to recover within days or weeks to an apparently asymptomatic state. A t this time the cholinesterases of blood plasma and erythrocytes w i l l usually exhibit only a small fraction of their normal activity. F r o m knowledge of the relationship between the activity of the enzymes in blood and central nervous system and the recovery rate of the latter in experimental animals, it is safe to assume that cholinesterase activity in nerve tissue of these human subjects is also low after the acute symptoms have subsided. In contrast with the picture of acute poisoning, it is possible for per sons who are continuously exposed to organophosphorus pesticides to absorb the material so slowly and at such a uniform rate that acute symptoms are minimal or absent, even though cholinesterase activity in the blood, measured by accepted methods, is markedly depressed. Ganelin (5) has assembled an impressive number of such cases among persons occupationally exposed to organophosphorus compounds. Summerford et al. (11) have pointed out that the rate of change of enzyme activity appears to be as important as the degree of depression in determining whether an acute phase of the poisoning syndrome occurs. There has been much speculation concerning the mechanisms re sponsible for adaptation, and numerous suggestions have emerged. One

In Organic Pesticides in the Environment; Rosen, A., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1966.


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of these, which proposes that excitable tissues can acquire tolerance to abnormally high levels of acetylcholine, has recently received experimental support. Brodeur and DuBois (3) have reported that the free acetylcholine content of the brain of rats remained above control levels throughout a 60-day period of daily Di-Syston injections although the acute symptoms which followed each dose became progressively less severe after 8 to 12 days. Animals thus resistant to the pesticide were reported to be less susceptible to the cholinergic drug, carbachol. Our study was initiated because the mechanism of adaptation is still obscure, and much additional information is needed concerning the relationship between cholinesterase activity and the acetylcholine content of the central nervous system during the adaptation process. W e also desired to investigate the possibility that adaptation might involve compensatory changes in the production or release of neural mediators, other than acetylcholine, capable of modifying the effects of high levels of the latter within the central nervous system. Norepinephrine was the first such mediator selected for study. Materials and Methods

Female white rats obtained from Holtzman ( Madison, Wise. ) were used. The animals were held in our animal quarters for 2 weeks after their arrival. They were maintained on a diet of Purina Micro-mixed Laboratory Chow, fed ad libitum, and weighed approximately 190 grams each at the beginning of an experiment. The experimental animals received daily injections of Di-Syston, 1 mg. per kg., given intraperitoneally. The pesticide was dissolved in a solvent mixture consisting of 10 parts of ethanol and 90 parts of propylene glycol, in a concentration of 1 mg. per ml. Control animals received daily injections of an appropriate volume of the solvent mixture, at approximately the same time each day. Di-Syston was selected as the cholinesterase inhibitor for use in this study because of a suitable duration of action and because the acutely toxic but nonlethal dose has been established ( 2 , 3 ) . Preliminary tests proved that its ethyl groups do not interfere with the gas-chromatographic estimation of acetylcholine. One or more animals was taken from the experimental group for brain acetylcholine measurement after 3, 10, 17, and 24 Di-Syston injections. In each instance, 24 hours had elapsed after the respective injection had been given. Cholinesterase activity of blood plasma, erythrocytes, and brain, and the norepinephrine content of brain were measured 24 hours after the third, tenth, and 24th injections. A l l of these measurements were repeated on the 38th day, 14 days after the last administration of Di-Syston. Animals were taken from the control group at each of the sampling times for the measurement of normal values. Each constituent was measured in the whole brain of an individual animal. A l l animals, control and experimental, were weighed daily through the i n jection period, and again at the 38th day.


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The entire experiment was repeated three times, consecutively, each time on a newly purchased group of animals. In each instance the starting ages and weights were nearly identical. Animals taken from experimental or control groups for the analytical procedures were decapitated by guillotine. The brain of each animal was then removed as rapidly as possible, the total elapsed time seldom exceeding 20 seconds. Further processing of the tissue depended upon the constituent to be measured. Acetylcholine. Acetylcholine was measured by a new gas chromatoDownloaded by NORTH CAROLINA STATE UNIV on May 4, 2015 | http://pubs.acs.org Publication Date: June 1, 1966 | doi: 10.1021/ba-1966-0060.ch006

graphic method developed in this laboratory (10).

Use of this method

in the measurement of tissue levels of acetylcholine has been described in detail (9).

Briefly, the tissue is frozen in a dry ice-ether bath imme-

diately after excision, weighed, crushed, and extracted by a modification of the method of Crossland (4)

with an acetic acid-alcohol mixture.

After centrifugation and washing of the residue, the supernates are combined and evaporated to dryness under reduced pressure.

The residue

from the evaporation is taken up in water acidified with acetic acid and the product is centrifuged.

Again, the supernate is evaporated to

dryness. The residue is dissolved in a small volume of water, and solid potassium borohydride and calcium chloride are added. The subsequent reaction converts the acetate moiety of acetylcholine to ethanol, which is then assayed by means of a gas chromatograph. The method is essentially specific for alcohol esters of choline and measures the total acetylcholine (free plus bound) content of the brain tissue. There is no interference from other substances which might have acetylcholine-like effects in biological assay.

The results are expressed

as micrograms of acetylcholine per gram of wet tissue. Cholinesterase A c t i v i t y . Cholinesterase activity was assayed by automatic, continuous, alkali titration of acid released from the substrate, a method previously utilized by Wilson (12),

M a i n and Dauterman

(6),

Shellenberger et al. (8), and others. Our procedure employed the Metrohm pH-Stat system in which the reaction proceeds at constant temperature and p H in an unbuffered, balanced-ion medium calculated to afford optimal conditions for enzyme activity. The closed reaction vessel is preflushed with nitrogen. In this inert atmosphere there is no interference from atmospheric C 0 , and because there is no spontaneous hydrolysis of the substrates during the short reaction period, blank runs are unnecessary. Only enough substrate is added to allow a linear reaction for a maximum of 10 minutes at the highest activities encountered, so that substrate inhibition is minimal. A description of our final version of this method, which is now being standardized on human subjects, will be published elsewhere. Cholinesterase activity of blood and brain was measured in the same animals. Blood was collected in heparinized tubes at the time of decapitation. Plasma and cells from 2 ml. of blood were separated imme2



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diately by centrifugation. The hematocrit value was obtained on a separate sample. Butyrylcholine iodide monohydrate ( Calbiochem grade B ) was used as the substrate for plasma assay, and the results are expressed as micromoles of butyric acid produced per milliliter of plasma per minute. Acetylcholine perchlorate was used as the substrate for erythrocyte and brain cholinesterase. The washed erythrocytes from 2 ml. of blood were diluted with the solution mentioned above, to which saponin was added to lake the cells, and an aliquot was taken for assay. The results are expressed as micromoles of acetic acid produced per minute per m i l l i liter of whole blood. Because we preferred to express erythrocyte activity in terms of whole blood, we adjusted each final value to its equivalent at a hematocrit of 5 0 % . This calculation, involving the hematocrit obtained on each blood sample, serves to differentiate enzyme inhibition from the variability in activity associated with abnormal plasma-erythrocyte volume ratios. The brain was homogenized in the saline diluent which is used in the assay procedure, and an aliquot of uniformly suspended homogenate was transferred directly to the reaction vessel. The results are expressed as micromoles of acetic acid produced per minute per gram of wet tissue. Norepinephrine. The norepinephrine content of the whole brain was measured by the method of Anton and Sayre ( J ) . Results

After the second injection of Di-Syston, the animals began to lose weight and continued to do so for 7 days. After the 7th day they began to gain weight, and for the remainder of the injection period their growth curve roughly paralleled that of the control group. W h e n the administration of Di-Syston was discontinued, the gain in weight accelerated, and at the 38th day, 14 days after the last injection, the animals remaining in the two groups weighed approximately the same. These weight relationships are illustrated in Figuré 1. Signs of poisoning reached their peak at about the 3rd day. They consisted of diarrhea, tremor, hyperexcitabihty, and excessive secretions from eyes, nose, and mouth. After the 3rd day the severity of the objective signs began to diminish, and by the 10th day they had subsided to a mild tremor and a slight but clearly apparent muscular weakness. Although these latter signs persisted until the injections were discontinued, the daily injections failed to elicit the acute phase seen earlier. Table I shows that the cholinesterase activity in the brain 24 hours after the third Di-Syston injection was approximately one third of the control value. Erythrocyte cholinesterase activity at the same time was inhibited to about the same degree, but the activity of the plasma esterase was only 2 5 % of the control level. During the succeeding 21 days of the injection period the brain cholinesterase activity declined further and remained at the 2 0 % level while the activity of the plasma enzyme i n -


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260 r

120

L-

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0 Figure 1.

«

ι

I

I

I

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ι

4 8 12 16 20 24 28 32 36 DAYS AFTER FIRST DI-SYSTON INJECTION

I

40

Effect of chronic Di-Syston administration on body weight

creased slightly and paralleled erythrocyte activity at about one third of the normal value. Fourteen days after the last Di-Syston injection of the series, brain and erythrocyte cholinesterase activities had returned to 66 and 6 0 % of normal, respectively, and the activity of the plasma enzyme exceeded the control level. Figure 2, A, shows the response of brain cholinesterase to the daily Di-Syston injections, expressed as percent of the activity measured i n control animals sacrificed on the same days. The curve was derived from Table I. Cholinesterase Activity of Rat Tissues Days after Brain, Erythrocytes, Plasma, First Di-Syston ^moles Acetic μmoles Acetic pmoles Butyric Injection Acid/G./Min. ± SE Acid/Ml /Min. ± SE " Acid/Ml/Min. ± SE Di-Syston 3 10 24 38 a 6 c

c

2.50 ±0.14 (4) 1.50 ±0.27 (5) 1.40 ±0.19 (5) 4.25 ±0.12

Control (3) 7.30 ±0.05 (2) 7.35 ±0.07 (2) 6.80 ±0.04 (2) 6.40 ±0.02

Di-Syston (6) 0.38 ±0.03 (4) 0.38 ±0.02 (5) 0.58 ±0.05 (5) 1:03 ±0.09

Control (3) 1.05 ±0.05 (2) 1.30 ±0.03 (2) 1.88 ±0.00 (2) 1.73 ±0.20

Di-Systo?i (5) 0.20 ±0.02 (4) 0.30 ±0.03 (5) 0.30 ±0.00 (5) 1.35 ±0.05

Control (3) 0.80 ±0.03 (2) 1.00 ±0.33 (2) 0.90 ±0.00 (2) 1.10 ±0.30

Per ml. whole blood adjusted to 50% hematocrit. Numbers in parentheses represent number of animals used. Estimations made 14 days after last injection of Di-Syston.



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data given i n the first column of Table I, i n which the standard error of each mean value is indicated. It is presented adjacent to the acetylcholine curves to show more clearly the quantitative relationships between the two sets of measurements. Although there was appreciable variability in the cholinesterase activity of the brain from animal to animal, we are confident that the percentages shown are reliable and that the form of the curve is accurate. Figure 2, B , presents the most significant results of this study i n graphic form. It shows that the acetylcholine content of the brain rose from a control level of 3.5 /*g. per gram of tissue to 4.67 fig. per gram 24 hours after the third Di-Syston injection. It had returned to 3.57 /xg. per gram after the tenth injection and remained at or near the control level thereafter. Table II shows that the norepinephrine content of the whole brain of animals receiving Di-Syston did not vary significantly from that found in control animals at any time during the injection period, and was precisely at the control level 14 days after administration of the pesticide had been discontinued.

Effects of Chronic Poisoning by an Organophosphorus Cholinesterase

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