Camp. Biochem. Physiol. Vat. 92C, No. 2, pp. 231-235, 1989 030&4492/89 $3.00 + 0.00 Printed in Great Britain 0 1989 Pergamon Press plc

CORRELATION OF THE STRUCTURES OF DIETHYLPHOSPHATE PHENYLESTERS TO HOUSEFLY ACETYLCHOLINESTERASE INHIBITION, THEIR SIDE

EFFECTS AND TOXICITY

TAKASHX ABE, YASUKO YAMADA and JUNICHI FUKAMI

Laboratory of Insect Toxicology, Inst;tut~ of Physical and Chemical Research, Hirosawa 2-1, Wako-shi, Saitama, Japan

(Received 7 March 1988)

Abstract-l. Diethylphosphate esters of o-, M-, and p-cresols, o-, M-, and p-nitrophenols, Znitro-p-, 4-nitro-m- and S-nitro-o-cresols, thymol, cateehol, m-xylenol, a and B naphthols, cyelododecanol and 3,4,5-trichlorophenol showed toxicity forward the housefly and inhibited housefly acetylcholines~r~.

2. The negative charges in the phenol moiety particularly in nitro, cyano and chloro groups, indicated low lr,, and LD~,, topically and injected.

3. In the housefly, the LD~ ratio of topical application to injection (d), which is characteristic of cuticular detoxication, was logarithmically correlated to 1% (r = 0.869) during a range of 1% concentration less than 2 x 10-4M.

4. The enzymic inhibitors containing nitro and cyano groups generally showed a low penetration, otherwise greater detoxication of the cuticle.

5. The toxicity of all the synthetic compounds examined was closely correlated to d (cuticular detoxication) (r = 0.866). Each isomer distributed accompanying with the regression line.

6. The isomer compounds were closely related to cuticular detoxication and toxicity. 7. The ratio of LD~ injected to 1% (s), i.e. the dependency of toxicity against acetylcholinesterase

inhibition, multiplied by d gave a constant. In case of this equation was graphically plotted on logarithmic scale, the value of S was given as - 1.26D - 2.63 (r = 0.959).

8. It thus appears that higher reactivity introduces cutieular detoxication and side effects of the compounds.

INTRODUCTION

It is well known that the outer skeleton of an arthropod contains a cuticle which prevents the pen- etration of ionic compounds as well as water (William, 1984). This is the means that insects have acquired through evolution to ensure that they are completely water proof for terrestrial life. The outer surface of an insect cuticle is covered by a wax layer (Hadley, 1980) with a high affinity for oily substances but not for hydrophilic compounds. Thus, it is gener- ally considered that hydrophobic compounds are more permeable than the hydrophilic or ionic com- pounds (Bocash et al., 1981; Tuli and Mehrotra, 1980). Such penetration follows first order kinetics both theoretically and in actuality (Buerger, 1967; Treherene, 1957). Cuticular ~~eability is the domi- nant factor in the selective toxicity of many insecti- cides; in fact, the non-ionic compound is highly toxic to an insect, but shows only slight toxicity to mam- mals (Winteringham, 1969). On this basis, many pesticides have recently been developed to be hydro- phobic. However, the question arises as to whether the pesticidal action really depends on affinity or penetration of the cuticle.

The toxicity of organophosphates has been found to be directly related to the inhibition of acetyl- cholinesterase. In our previous paper, it has been shown in several diethylphosphate phenylesters that phenol derivatives with negative charge (as a nitro

group) strongly inhibit acetylcholinesterase activity (Abe et al., 1984).

However, many studies, including our earlier work, have indicated many side effects from these com- pounds such as that of decoupling on respiration by p-nitrophenol decomposed from paraoxon (Parker, 1958), muscular membrane depolarization by para- oxon (Abe et al., 1983) and blocking accompanied by spontaneous repetitive excitation of aminergic neuro- muscular preparation by several organophosphates (Abe ef af., 1981, 1988) or repetitive excitation of cholinergic neuromuscular junction by paraoxon (Clarke et al., 1980, 1984). These chemicals and metabolites possibly inhibit not only acetyl- cholinesterase activity but many physiological pro- cesses as well. In this paper, an attempt was made to determine the manner in which total toxicity, cuticu- lar penetration, acetylcholinesterase inhibition and side effects are correlated to the structures of various diethylphosphate esters.

MATERIALS AND METHODs

~uteriais

Cyclododecanol and catechol were purchased from Tokyo Kasei Chemical Co. and Daiichi Chemical Co., respectively. Diethylchlorophosphate was provided through the courtesy of Aldrich Chemical Co. Acetylthiocholine iodide and dithio-bis-nitrobenzoic acid (DTNB) from Kanto Chemical Co. and Sigma Chemical Co., respectively.

231 C.B.P. ‘WC--E

232 TAKASHI ABE er al.

A Sephadex G-100 of Pharmasia was used for the gel filtration.

Method

Synthesis of diethylphosphate phenylesters. In our previous paper, we synthesized several phosphotriesters and showed their physico-chemical properties (Abe et al., 1984, 1988). In the present study, phosphotriesters of catechol and cyclo- dodecanol were synthesized by same method as described previously (Abe et al., 1988).

Physical analysis of the synthetic compounc&. Infrared and NMR spectra, and elemental analysis conducted using the same procedure as that in our previous papers (Abe et al., 1984, 1988) are listed in Table 1.

Toxicity test. Prior to topical application of the chemicals, houseflies of the SRS strain were anesthetized by CO,. Each chemical, after being dissolved in 1.0~1 of acetone, was applied to the dorsal thorax of houseflies. The injections were performed on the dorsal thorax after dissolving the chemicals in 1 ~1 of 30% acetone. Three replicate tests using 20 flies at each of five concentrations of the chemicals were carried out. Mortalities were calculated for 24 hr at 25°C following administration. Median lethal doses (LDSo) were determined by probit analysis.

Other methods. Preparation of housefly acetylcholinester- ase and inhibitory study of the enzyme were carried out according to previous methods (Abe et al., 1984). The acetylcholinesterase was assayed by the method of Ellman et al. (1961).

RESULTS

Correlation between structures of synthetic compounds and inhibition (rsO) of housejly acetylcholinesterase

All the synthetic compounds were found to inhibit housefly acetylcholinesterase activity as shown in Table 2. The phenol structures of some of the chemi- cals had negatively charged nitro, chloro and cyano groups. Such groups may possibly be indispensable to a strong inhibition of enzymic activity and increase molecular hydrophilicity and polarity (Fukui et al., 1961). In spite of its hydrophilic structure, the catechol derivative failed to markedly inhibit the activity of this enzyme. Thus the negative charge, rather than hydrophilicity, is the factor responsible for the strong inhibition of the enzyme. A compara- tive study of the nitro groups on the phenol and cresol structures indicated the para position to inhibit activity more efficiently than the metha or ortho position.

In contrast, compounds containing a hydrophobic group in the phenol moiety such as a methyl or aromatic group were approximately 100 times less inhibitory than those with negative groups. It thus appears that the value of I,, decreases either the degree of electrophilicity of the substituted residues, or hydrolizability of the compounds. This possibly may be due to superdelocalizability of P atom or stability of the ester bond (Fukui et al., 1961).

Toxicity of the synthetic compounds

The toxicity toward the houseflies was analyzed by LDSO injection and topical application. The LD, topi- cally was about 100 times that of the LDso injected. The value of former was closely related to that of latter (r = 0.843). For each compound, both values were correlated to the I~,, of acetylcholinesterase (r = 0.956 for LD~~ injected and r = 0.825 for LD5,,

Housefly AChE inhibition 233

Table 2. I~ of housefly acetylcholinesterase and LOO of housefly for diethylphosphate phenylesters

Derivatives of diethylphosphate

1. 0 -cresol 2. mcresol 3. p -cresol 4. o-nitrophenol 5. m-nitrophenol 6. p-nitrophenol 7. 2-nitro-p-cresol 8. 5-nitro-o-cresol 9. 4-nitro-m-cresol

IO. thymol 1 I. catechol 12. m -xylenol 13. a-naphthol 14. b-naphthol 15. cyclododecanol 16. p-hydroxybiphenol 17. p-cyanophenol 18. p-chlorophenol 19. 3,4,5-trichlorophenoi

LD,(rnOl)

(N-tllOl) Topically Injected

340 3.00 x 10-7 2.69 x lO-9 600 2.00 x 10-h 1.80 x IO-’

1380 9.60 x IO--’ 5.15 x 10-g 12.0 5.92 x IO-’ 2.42 x IO-‘*

7.60 1.74 x 10-9 1.50 x lo-‘* 2.84 7.66 x IO-” 1.69 x lo-‘* 3.10 3.00 x 10-a 4.80 x IO-‘”

247 4.50 x IO_@ 3.55 x IO_” 0.168 2.09 x 10m9 6.30 x IO-”

182 2.70 x IO-’ 1.39 x 10-8 4500 3.65 x W9

43.2 7.66 x IO-’ 6.50 x 10mp 335 7.00 x IO_ ’ 6.70 x 10mp

1040 5.40 x 10-7 1.66 x 10-g 10.9 8.10 x IO-’ 1.30 x 10-g

124 . 2.34 x IO-’ 1.51 x 10-8 10.9 7.20 x 10m9 7.60 x 10m’z 46.0 1.80 x IO-” 4.95 x W’O

0.0575 2.96 x 10mq 8.70 x 10m”

topically). Esters containing nitrophenol, nitrocresol, chlorophenol and cyanophenol usually showed low I~,, and LD~. However, higher values were obtained for nonpolar molecules (see Table 2). Since nonpolar molecules can permeate the cuticle more easily than polar molecules (Bocash et af., 1981, and the data of the present study), LD5,, may possibly not be influenced much by cuticular penetration but rather by enzymic inhibition.

Acetylcholinesterase inhibition and cuticular detoxication

The ratio of LDSO values from topically application and injection (d) indicates how many times topical application must be made in order to have the equivalent effect of the injection for lethal dose. It indicates penetrative inhibition of the cuticle, since effective penetration possibly depends on diffusion of the lethal amount minus cuticular detoxication which includes adsorption and decomposition of active compounds. The approximate degree of cuticular detoxication can be determined from this ratio. A decrease in d may result in either an increase in cuticular detoxication or a decrease in cuticular pen- etration. Thus, the cuticular detoxication will depend on labile property or affinity of the chemicals against cuticular materials.

When the d and i (the value of I~,,), were plotted on a logarithmic scale (D and I), it showed a negative correlation D = - 1.2181- 3.24 (r = -0.869) was obtained (see Fig. 1). Many effective inhibitors showed a higher D value and a linear relationship, except extremely high I~,, At over 2 x 10e4 mol of I~,,, D randomly increased since LD5,, topical application was amplified rather than the LDSO injected. It is suggested that the compounds with strong inhibi- tory action (low I~~) may possibly easily undergo detoxication on the cuticle.

Cuticular detoxication and toxicity

The correlation of cuticular detoxication (d) to

LDso injection (t) was actually plotted on the logarithmic scale of the linear equation of D = -0.510T - 2.55 (r = -0.866) (see Fig. 2). This

equation indicates the increase in toxicity to possibly result from that in cuticular detoxication or decreased penetration.

From Fig. 2, it appears that isomers of the com- pounds are closely related to cuticular detoxication (d) and toxicity. By the structural correlation, those isomers may have similar properties with respect to cuticular detoxication, the metabolism of detoxi- cation and inhibitory activity in the insect. As dis- cussed below, the cuticle was found to function as a filter of chemically active compounds, and to be less porous to active chemicals.

Acetylcholinesterase-dependent toxicity (s): cuticular detoxication (d) plot of the synthetic compounds

The ratio of LDSO injected to I~,, (s) indicates the dependency of the toxicity against acetyl-

01 , -7 -6 -5 -4 -3

I (Iso)

Fig. 1. Correlation of acetylcholinesterase inhibition to cuticular detoxication. Numbers are chemicals listed in Table 2. At over 2 x 10m4 mol of I~, the linear relationship was not observed by excess increase of LD~ topically. The regression line showed a function of D = - 1.2181- 3.24

(r = -0.869).

234 TAKASHI ABE et al.

s! 43 -12 -11 -10 -9 -e -i T (LDso Inij

Fig. 2. Relationship of toxicity to cuticular detoxication. Numbers are chemicals listed in Table 2. A function of the regression line were D = -0.510T - 2.55 (r = -0.866). The isomer or similar compounds were linked by dotted

line.

cholinesterase inhibition. It was especially low in compounds containing nitro and cyano groups. This ratio increases when chemicals are easily detoxicated on the cuticle, resulting in extremely high value of LD~,, injected or the low I~~. At low I,,, it decreases and the chemical shows various inhibitory effects, i.e. side-effects in addition to acetylcholinesterase inhibition.

Both acetylcholinesterase-dependent toxicity and cuticular detoxication were correlated on an equation of s x d = a constant. This equation shows the cut- icular detoxication to be inversely proportionated to acetylcholinesterase-dependent toxicity. When each of these values for the synethetic compounds was logarithmically plotted on a graph, the function, S = - 1.26 D - 2.63 (r = -0.959) was obtained. The slope coefficient indicates the inhibitory strength of the side effects. The equation shows that cuticular penetration and side effects are inversely propor- tional. Actually, compounds with greater cuticular detoxication caused side effects, such as respiratory decoupling (Parker, 1958), muscular membrane de- polarization (Abe et al., 1983), blocking accompanied

Fig. 3. Aeetylcholinesterase-dependent toxicity: cuticular detoxication plot of the diethylphosphate phenylesters. The regression line except chemical Nos 7, 9, 12 and 19 was

S = - 1.260 - 2.63 (r = -0.959).

by spontaneous MEPPs of aminergic neuromuscular preparation (Abe et al., 1981, 1988), and repetitive response of cholinergic neuromuscular junction (Clark et al., 1980, 1984), without acetylcholinester- ase inhibition.

There were two isomer groups which deviated from the D-S rule. One group consisting of Nos 18 and 19 contained chloride in the phenol ring. Both had similar D-values, but the S-value of No. 19 tended, remarkably, to increase by strong inhibition of acetyl- cholinesterase. The other group, consisting of Nos 7, 8 and 9, had similar values for S, but the D-value of Nos 7 and 9 increased much more than that of No. 8. These findings indicate possibly high toxicity and greater selective toxicity of the inhibitors for the two groups, respectively. In a series of compounds adher- ing to the S-D rule, high reactivity or impermeability caused the side-effects.

DISCUSSION

In a previous paper (Abe et al., 1983), the presence of a nitro group in the phenol ring of diethyl- phosphate ester was found to enhance the inhibition of acetylcholinesterase. The experiments conducted in the present study demonstrated negative charges in the aromatic ring to generally be necessary for strong inhibition. These charges increase also the hydro- philicity of the inhibitors, but a derivative of catechol which is hydrophilic failed to strongly inhibit the enzymic activity. Thus the negative charge might be a very important factor for enzymic inhibition. In contrast, hydrophobic derivatives decreased inhibi- tory activity possibly due to their low solubility in the reaction mixture. However, both topically applied and injected LD5,, increased proportionally as did I~. It thus appears quite likely that the inhibitory site of housefly acetylcholinesterase has affinity for negative charge, even though a hydrophobic site of this enzyme has been suggested in several reports (Becker ef al., 1963; Bracha and O’Brien, 1968a, b). The negative charge of inhibitors also possibly enhances the hydrolyzability of the ester bond and causes phosphorilation of the catalitic site.

On the expression of toxicity, cuticular penetration by insecticides is an important factor determining the extent of lethality. The lethality of compounds for practical use is thus considered to depend on their affinity for the cuticle. The insect integument is com- prised basically to epicuticle, exocuticle and endo- cuticle. The surface of the epicuticle is covered with wax produced on epidermis and secreted to the surface through a wax canal. Thus hydrophobic compounds having good affinity for this wax should permeate the chitin of the lamella structure or be- come incorporated into epidermis through the wax canal more easily than hydrophilic substances. In this experiment, the hydrophobic compound was found to depend strongly on cuticular permeability (Figs 1 and 2). The hydrophobicity of a compound is the primary quality necessary for cuticular penetration, but is not the dominant factor for the insecticidal activity (Table 2).

The cuticle generally prevents penetration of substances applied topically as described in the Introduction. As shown by the data presented above

Housefly AChE inhibition 235

(see Table 2), a large amount of the chemical applied was detoxicated by decomposition and adsorption on cuticle. The small amount remaining penetrated and infused the body of the insect. Accordingly, the amount of topically applied LD~,, that penetrated the insect body is nearly the same as the amount of injected LD% . The ratio of topically applied and injected LDSO (d) shows therefore cuticular detoxi- cation compared to cellular toxicity, and is an index of cuticular defence. The other hand, the cuticular detoxication or the impermeability increased propor- tionally to acetylcholinesterase inhibition and toxicity as shown in Figs 2 and 3. This may possibly be based on both properties of cuticle and chemicals. Non- polar hydrophobic compounds have a good affinity on the wax layer of hydrocarbon (Hockman, 1974). However, polar hydrophilic compounds show a good affinity on protein epicuticle (Pfaff, 1952). When it has been considered that the acetylcholinesterase inhibition and the side-effects are fundamentally de- pendent on the affinity or the binding to enzymes and functional proteins, we understand that the nonpolar hydrophobic organophosphates penetrate first and have a low toxicity, contrary to the polar hydrophilic compounds which inhibit the enzymes and suppress the penetration.

Tenebrio molitor by phosphotriesters. Comp. Biochem. Physiol. (in press).

Becker E. L., Fukoto T. R., Boone B., Canham D. C. and Boger E. (1963) The relationship of enzyme inhibitory activity to the structure of n-alkylphosphate and phenyl- alkylphosphate esters. Biochemistry 2, 72-76.

Bocash W. D., Cornford E. M. and Oldendorf W. H. (1981) Shistosoma mansoni: correlation between lipid partition coefficients and the transintegumental uptake of non- electrolytes. Exp. Parasit. 52,369~403. _

Bracha P. and O’Brien R. D. (1968a) Trialkyl phosphate and phosphorothiolate anticholinesterases: I. Amiton analogs. Biochemistry 7, 1545-1554.

Bracha P. and O’Brien R. D. (1968b) Trialkvlnhosohate and phosphorothiolate antichohnesterases: II: Effec& of chain length on potency. Biochemistry 7, 1555-1559.

Buerger A. A. (1967) A theory of integumental penetration. J. Theor. Biol. 14, 66-73.

Clark A. L., Hobbiger F. and Terrar D. A. (1980) Intra- cellular recording of the anticholinesterase-induced repetitive responses of mammalian muscles to single indirect stimuli. J. Physiol. 302, 26P-27P.

Clark A. L., Hobbiger F. and Terrar D. A. (1984) Nature of the anticholinesterase-induced repetitive response of rat and mouse striated muscle to single nerve stimuli. J. Physiol. 341, 157-166.

The S-D rule shows that the high reactive com- pounds possibly react with the cuticular and cellular components to inhibit permeability and enhance cellular toxicity, respectively. The intensity of side effects increases with the individual reactivity of several compounds. Expression of this fundamental property should occur for any chemical used and regardless of the species of insect or the resistancy for chemicals.

Ellman G. L., Courtney K. D., Anders V. Jr and Feather- stone R. M. (1961) A new and rapid calorimetric determination of acetylcholinesterase activity. Biochem. Pharmac. 7, 88-95.

Fukui K., Morokuma K., Nagata C. and Imamura A. (1961) Electronic structure and biochemical activities in diethyl phenyl phosphates. Bull. them. Sot. Japan 34, 1224.-1227.

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