蛔蟲體內 cholinesterase活性의 所在와 有機燐劑의 抑制作用에 關한 組織化學的 硏究

Abstract

Introduction

Concerning Cholinesterase (Ch.E) activity in the body of parasites, Heeding (1952) revealed its existence in Schistosoma mansoni. Lui et al. (1964) reported that acetylcholinesterase (Ac7E) activity in the body of Ascaris suum was recognized mainly in the anterior part of the body as well as the alimentary duct.

Beiejac (1964) also found activity from the sucker, ganglia and longitudinal nerve fibres of Dicrocoelium lanceatum. Knowes and Casida (1962) reported that Ch.E activity in Ascaris lumbriccideswas inhibited by Haloxon [di-(2-chloroethyl)

3-chloro -4- methyl -7- coumarinyl ohosphate] but it recovered within 24 hours. But Hart and Lee (1966) found that Haloxon sometimes resulted in a permanent inhibitory action upon AchE activity in Ascaris.

Dipterex (o, o-dimethyl 2, 2, 2-trichloro -1- hydroxyethyl phosphonate) has been recognized as a low toxic organophosphorus compound. Cerf et al. (1962) reported that it was less toxic to the host and highly effective in eliminating Ascaris lumbricoides. Recently, Kahn et al. (1967) did a follow-up study with the chemical and found it had about 75% effectiveness in eliminating Ascaris. However, there has been no study concerning the mechanism of its anthelmintic activity.

The present study was designed to investigate the anthelmintic action of the organophosphorus compound by examining the AchE activity in the tissues of Ascaris in human hosts.

Materials and Methods

Materials:

(A) Parasites

Two Ascaris lumbricoides without receiving anthelmintics were obtained from the intestine during surgical operation. But the other warms, in accordance with the purpose of the experiments, were obtained by administering several drugs (Santonin, Kainic acid, Piperazine and Dipterex) to the boys and girls infested.

(B) Chemicals and drugs.

Inhibitors : Eserine (Merck)

Dipterex (Bayer)

Substrate : Acetyl-thiocholine iodide (U.S.P.)

Anthelmintics : Dipterex (Bayer)

Piperazine

Kainic acid (JP)

Santonin

Methods:

a. Two living Ascaris (untreated) were kept in saline (at 35"C. for 1 hr.), Then one was cut into three pieces from mouth part, central part, and tail part the length of 1 cm each and fixed in formalin sucrose ammonia solution (0"C). (The method of section and fixation were the same in the following experiments). The

other worm was put into a beater containing 300 cc of Dipterex solution.1diluted to 2000X and retained in incubation at 35℃ for 24 hours. Then three similar suctions were made and fixed in the fixative.

b. Three living Ascaris lumbricoides were obtained by giving Santon in (0.1 gm) to the boys infested(15-16 in age). The worm? were washed several times with warm saline solution. and retained in saline solution at 35℃ for 24 hours. Then, each was Put into beakers containing 30? co of Dipterex solution diluted to 1000X, 2000X and 3000X respectively, The beakers containing Ascaris were incubated (35"C) for 10 hrs. Then sections were made and fixed respectively.

c. Four Ascaris lumbricoides expelled by dosing with various anthelmintics: Santonin (0.1 gm), Kainic acid (10 gm), Piperazine citrate (1.5 gm) and Dipterex (15 m7/k7, 2 d7s73 with 24 hours. interval) were washed several times with warm saline solution. Then m37e sections were and fixed respectively.

d. Koelle's method was selected for histochemical staining.

e. Eserine test was performed with 10-s M as used by Pearse (1967) prior to incubation in the substrate.

Reaults

A. Ascaris lumbricoides (Control, not treated with chemical): The histochemical view obtained from mouth part, central part and tail part were as follows:

(1) Cuticle: The cuticle consisted of two spiral cards. One had dark brown stain but the other no stain reaction. Above was observed from all three portions.

(2) Body wall: The round muscle cells showed no enzyme reactions. The border of muscle tissue facing coelom was stained dark brows. This was observed from central and tail part, but only slightly in the mouth part.

(3) Alimentary duct: On the outer edge of the muscular tissues, dark brown stains formed a thick circulation around the alimentary tract, and diverse reaction spots were observed from the periphery of this area. Spindle epithelia were not stained

but slight darkbrown stains were observed along epithelial membrane facing the canal. This was observed in the central and tail parts, but meagerly in the mouth part.

B. Eserine abolished the stain reactions distributed at large in the body of normal A. lumbyicoides.

C. Histochemical appearance of Ascaris lumbricoides reacted by Dipterex solution (1000X, 2770X,3000X, diluted) for 10 hours. in vitro.

(1) Cuticle: Ch.5 activity localized in the spiral cord was abolished in 1000X, 2000X and 3000X diluted segments.

(2) Body wall: Ch.E activity on the border of muscular bundles was present throught the segments of 1000X, 2000X and 3000X dilution.

(3) Alimentary duct: Ch.B activity on the outer edge of muscular tissue disappeared in the segment of 1000X dilution. It was slightly diminished in the muscular tissue in the segmea of 2000X dilution. No influence of Ch.E activity was'-recognize? in the segment of 3000X dilution.

D. Retraction of Dipterex solution (2000X) to Ascuis lumbricoides in vitro.

(1) Cuticle: The dark brown staining of the spiral cord was abolished.

(2) Body wall: The stain on the border of muscular bundles facing the coelom was absent.

(3) Alimentary duct: The massivc dark brown stain of the muscular tissue on the edge was inhibited totally as well as the diverse stained masses localized over the space of epithelia and the dark brown spots along epithelial membrane.

E. Ascaris lumbricoides expelled by dosing with several anthelmintics in vivo.

Three Ascaris lumbricoides expelld by dosing with Santonin, Kainic acid, and Piperazine showed the same appearance as the Ascaris lumbricoides controls. No change was found in the stain reaction, but Ascaris tumbricoides expelled by dosing with Dipterex gave a similar result as in "B" and"D" .

Summary

In order to study the anthelmintic mechanism of an organophosphorus compound (Dipterex) in Ascaris lumbricoides, the Ch.E activities in the parasites treated with the chemical were examined histochemically. The results were as follows:

(1) In normal A. lumbricoides, the stain reaction was observed on the spiral cord of the cuticle and in the connective tissue cells of the body wall layer. Particularly marked stain reaction was observed at the base of muscular tissue of the alimentary duct which would be considered the end plate of neuromuscular

junction.

(2) The stain reaction in A. lumbricoides incubated in Dipterex solution diluted 2000X for 24 hours was completely inhibited.

(3) The stain reaction in 7. lumbricoides from the human host was completely inhibited by dosing with Dipterex in vivo.

(4) The non-organophosphorus compounds (Santonin, Piperazine, and Kainic acid) did not inhibitCh.E activity in the bodies of A. lumbricoides.

Through above results, it is considered that the anthelmintic mechanism of organfphosphorus compound is due to the inhibitory action of Ch. E activity in Ascaris lumsricoides.