FISHERIES RESEARCH BOARD OF CANADA

Translation Series No. 1503

Toxicological and biochemical research of pesticides using radioisotopes.

By Junichi Fukami

.Original titl: RI riyo ni yOru Noyaku no Yakuri-to Seikagaki. U - '

From: Hoshseibushiteu (Radioisotopes), 18 (9): 385-401, 1969.

.Translated by the ,TranslatiOn Bureau (MI) , FOreign Languages Division - Department of the Secretary of State of Canada

Fisheries Research Board of Canada Freshwater Institute Winnipeg, Manitoba

1970-

68 pages typescript

TRANSLATED FROM - TRADUCTION DE INTO - EN

Japanese. English

PAGE NUMBERS IN ORIGINAL NUMROS DES PAGES DANS

L'ORIGINAL

PUBL ISH ER - EDIT EU R

no clue available to identify DATE OF PUBLICATION DATE DE PUBLICATION .

385 - 14 01 ISSUE NO. NUMRO

YEAR AN- NE

VOLUME PLACE OF PUBLICATION LIEU DE PUBLICATION

NUMBER OF TYPED PAGES NOMBFtE DE PAGES

DACTYLOGRAPHIES

18 no clue available to identify 9 1969 1 - 68

M. I .

28.7 .70

Fe-e /5 o3 DEPARTMENT OF THE SECRETARY OF STATE

.. TRANSLATION BUREAU

FOREIGN LANGUAGES DIVISION , CANADA

SECRTARIAT D'TAT BUREAU DES TRADUCTIONS

DIVISION DES LgANGUES TRANGRES

AUTHOR - AUTEUR

FUKAMI, Junichi

TITLE IN ENGLISH - TITRE ANGLAIS

Toxicological and Biochemical Research of Pesticides Using Radioisitopes Title in foreign language (transliterate foreign charaetera)

RI riyo ni yoru Noyaku no Yakuri to Seikagaku

REF5RENCE IN FOREIGN I,ANGUAGE (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATE FOREIGN CHARACTERS. REFERENCE EN LANGUE ETRANGERE (NOM DU LIVRE OU PUBLICATION), AU COMPLET.TRANSCRIRE EN CARACTERES PHONETIQUES.

possible)

Hoshaseibushiteu (or Hoshano bushitsu, Hoshasengaku)

REFERENCE IN ENGLISFI - RFRENCE EN ANGLAIS

Radioisotopes

REQUE.STING DEPARTMENT Fisheries & Fores try MIN ISTERE-CLIENT

TRANSLATION BUREAU NO. 1061 NOTRE DOSSIER NO

BRANCH OR DIVISION DIRECTION OU DIVISION

PERSON REQUESTING DEMANDE PAR

Fisheries Research Board

Dr. Jack Uthe, Freshwater institute. Winnipeg, Man.

TRANSLATOR (INITIALS) TRADUCTEUR (INITIALES)

DATE C.OMPLETED ACHEVE LE

YOUR NUMBER 769.-18-14. VOTRE DOSSIER N

DATE OF REQUEST 13.5.70 DATE DE LA DEMANDE

SOS-200-10.8 (R EV. 2/014

CANADA

Review Article /385

DEPARTMENT OF THE SECRETARY OF STATE

' eTRANSLATION BUREAU

FOREIGN LANGUAGES DIVISION

, f / 5-0 SECRTARIAT D'TAT

BUREAU DES 'TRADUCTIONS

DIVISION DES LANGUES TRANGRES

CLIENT'S NO. DEPARTMENT ' OIVISION/BRANCH cm, ' N9 OU CLIENT MINISTERE DIVISION/DIRECTION VILLE

769,- 18-14 Fisheries & Forestry. Fisheries .Research Board'Winnipeg Mari

BUREAU ND. L.ANGUAGE .TRANSLATOR (INITIALS)' MMrE No DU BUREAU LANGUE . TRADUCTEUR (INITIALES)

1061 Japanese . M.I... 29 Jule-1970

TOXICOLOGICAL.AND BIOCHEMICAL RESEARCH OF PESTICIDES USING'

RADIOISOTOPES -

. by .

Junichi FUKAMI

Laboratory of . Entomological Toxicology

Riken (Institute of Physical and Chemical Research)

Radioisotopes Vol. 18, No. 99 PP. 385-401 (1969)

Translator's Note: Table of Contents was added for clarity.- Some figures and chemical structures:were misprinted, and therefore they were rewritten, referring to:other chemical literature. The authOr mixed English terms, in phonetic writing ., .with German terms and they were translated into English. Some reference numbers in the text were misprinted, and by checking the authors' names, some could be . corrcted. A few could not be locate d . in the text,.while a few others were illegible (foot notes). The translator did obtain the author's address:.

Riken, YamatomaChi, Kitaadaphi, Saitama, Japan..

UNEDITED DRAFT TRANSLATION Orily for information

TRADUCTION NON REVISE .nforrnzCien soule.ment

SOS-200-10-31

II

(397)

(398)

.40.

CONTENTS

. . . . . Page (Original . . . . ..

1 introduction - 2 . (385)

. .

*. ". ' .2- Insecticides - 7 (386)

2.1 Insecticides ' . .. . . 7 . 0 ..

' 2:1.1 Rotencids . . 7

2.1.2 Pyrethroids- . 12 . (387)

2.2 OrganphOsphates 16 (388)

2.2.1 Exchange Reactions between S and 0

2.2..2 Oxidations of Sulfur

2.2,3 Hydroxylations of Alkyl Side Chains and N-dealkylations

2.3 Carbamate Insecticides

2.4 Organochloro Insecticides

2.5 Inductive Avtivation of Drug-oxidizing Enzymes by Organochloro Insecticides 36 (392)

3 Weedkillers 39 (393)

3.1 Trifluralin 40

3.2 Diphenamide 40

3.3 Diuron, Monuron 41

3.4 Dicamba 43 (394)

3.5 Paraquat, Diquat 44

3.6 Simazine, Atrazine 45

3.7 Propanil (

47 (395)

3.8 Naphthaleneacetic Acid 49

4 Fungicides 50

5 Selective TOxicity 56

5.1 Selective Tocicity of Rotenone 56

5.2 Selective Toxicity of Parathion Type Insecticides 60

Bibliography 65

., .22

., 24

.. 31

2 -

I. INTRODUCTION

The remarkable advances in developing various pesti-

cides together with the steady improvements of agricultural

technology in the recent years resulted in almost consecutive

increases of annual crops of rice and yields of other agri-

cultural products, particularly fruit and vegetables. The

consumption of pesticides in this.country also increased

enorMously in -recent years. In fact, the increment could e

. figured out from the difference in 'total output 's of pesticides,

*four billion Yens* .in . 1951 and Sixty-seVen billion and one

..hundred million yens* in 1967. Of these pesticides produced,

More than .90% of the products** ar.organic-chemically synthesized

compound. The pesticides comMonly used.during abd before the -

war*** were either natizral organic compounds such as - rotenoids

and. pyrethroids Or inorganic compounds', for example, - arsenic

chemicals. Consequently, nobody had shown interest in cumulative

or residual toxicity of the pesticides. However, many organic

synthesis products including DDT, BHC, parathion, 2.4-D,

organomercuric preparations and others became the more common

pesticides fter the war. While these synthetic organic

pesticides became popular, the unfavourable effects on the

general health of human beings also started to appear. These

*Translator's Note: 4,000,000,000 yens and 67,100,000,000 yens. 330 yens = 1 Canadian dollar.

** " " 90% of the kinds of product or of the total amounts?

*** " referring to W.W. II.

3 --

effects are indeed the . dark , side of the application -of

pesticides-, and thy'include i.e. the' poisoning .of users

of the pesticides, pesticide reeidties in the agricultural.

prOducts,,and . destruction of useful predatory'insects and.

animals. R.C. CarSon's "Silent Spring" (translatedinto

: Japanese" Sei to Shi no Myoya1u",.1962) 1). and the Yesner

, Report 2) of-America aroused the common intereSti_n the .

secondary . effects 0' pesticides,. emphasizing that in order

to reduce the unfavourable side effects of pesticides, safer,

and selective.methods of removing unwanted insects should .

be developd. They suggested that (a)*theuse of.selectively

toxic compotinds, (h) compounds Which did not leave residual -

matter, (c) application of methods which were selective in

use or' (d) the use of attractants and,cheMicals which inter-:

fered only with reproductiOn-, and further development of

methods whiCh did'not use ' any . chemical at all; might be.the

solutions. - In order to expioreHthese suggested methods, it

is.important.to study the mechanism.of the action of:pesti-:

ides, namely, their comparative toxicities, the metabbliems

in insects,. mammals, and Plante:1w- Understanding -Ole processes

. of the pesticides to *be . decompoeed and deactivated in nature :

is aleo one of the more important basic:problems to be studied.

In . gener1, newer methods.of removal of . insects are expected

to be derived from the resulte of the studies of baeic .

problems rather than from cumulating . experiences'only. These

studies shouId.aIso yield helpful:improvements and solutions

in removal of the insects which were rapidlibecoming resistant.

to the-existing.insecticides i. The 'author has been Working in

one of:thselasic problem areas, particularly the selective' . .

insecticidal aCtion of insecticides l *for . some.years. Although

their 'margins of selectivity are rather . wide, we - have already

found some insecticides which have a very low toxicity -fr

mammals and-destrby only the harmful insects. The diffrence

in the'activities of these insecticides - against insect and ,

maffimals is mostly depending.on . the qualitative and quantitative

differenCes of the metabolism systems of thee living'creatures.

TheMetaboiisms of the-pesticides'and other chemicals are, .

mainly, depending on the action of their ' enzymes. Therefore

the result of the, se enzyme actions - activation and deacti-

vation of pesticides --appear to yield the .width of the

margins of selectivitY of' the peticides. The firt step

in the metabolism'of the pesticides introduced into the body '

is'probablY oxidationreduction and hydrolysis, and the

second tep is the formation.of complexes of their primary .

.metabOlism products. The proceses - and the -mechanisms have

been explored mainly by using pesticides labeled mith radio

isotopes....This,author intends to ipreSent examples of

applications of radioisotopes mainiy'in the studies of-

oXidative metabolism,.which has been most .carefully studied

and further, explain their applications in hydrolysis and

*complex formation reaction. It wouldplease the author

greatly'if.this article could arouse the interest of those

who were engaged in the studie of the areas not directly

related to the pharmacology of insects.

As for th oxidation of the Chemicals, it:isWell

. known that tWo systems 'aire participating . in biological . ,

oxidations.one is - the enzyme system which oxidizes the

substrate in the mammalian liver microsomes in the presence

of NADPH* and pkygen, and the other i the system which includes -

.c.itoChrome P-450.. The chemicalsubtances, once introduced .

into the biological systms, are.oxidized by oxidation. enzymes, -

and the oxidation products further undergo Various complex.

, formations, for instance, acetylation, sulfonate 'ester

formation end gIucuronide formation. When fUnctional groups

that are 'harmful to biologiCaI metabolisms are masked .by

derivative formations, the deriVatiVes.are also - easily .brought

into.the excretory systems4'The major types of metabolisms -

which are carried out by the enzymatic .oxidations are (1) .

oXidation of alkyl side chains, - (2) hydroxylation of aromatic .

rings, (3) hydroxylation , cif non-aromatic rings, (4) dealkylation

ofN,alkyl compounds,. (5) dealkyIation of-Oalkyl compounds

(6) 'oxidation of amind - groups '(N-pxidation) (7) oxidation 'of

sulfur (sulfoxidation),..and (8).exchange reaction between -

*Translator's Note: Some 'authors use NADPH2 (reduced.nicotine adenine dinucleotide phosphate;'formerly TPNH). See also NADH in section 2.1.1. Some authors use NADH2 (reduced nicotine adenine dinucleotide; 'formerly DPNH).

S and O. Which type or .tyPes of oxidative metabolism take ...

place.when psticidesare . introduced - lnto biologial systems,

that is; the'selection of the type s. of metabolism, is not

. clearly understood. This difficult prediction is Mainly

because of the fact that the pesticides are applied against

many species of .mammals, fish, insect, plant and microbe,

and as. a result, just what kinds of oxidation enzyme exist

'in each species of living-creatures to oxidize certain kinds..

of pestiids is quite uncertain.. Even such a seeminglY simple

question as Whether eome species of insect's have - the - Same

kinds of oxidation enzyme as.foundin mammalian, liver micro-

somes has not been answered witn'reasonable - accuracy..Questions

such as this and the effort to answer the 'questions appear

. to .give the more vital drivingfprce and to orient the

direction in discovering the more .. .selective pesticides.

Dedigning of better qualified pesticides May be achieved /386

only by understanding the mechanism of the pesticidal . action

in each case Of applicatiOn.,

The author plans to explain the examples of application

of radioisotopes in the mechanism studies of insecticidal

actions, as this area is one of the most advanced of all the

pesticide studies, and . then to describe the studies in other,

areas in the order of weedkillers and fungicides.

. INSECTICIDES

2.1 Natural Insecticides r

. 2.1.1 Rotenoids

. Rotenone, the major component of derria root * , hae

been widely used'as a. natural insecticide -since before the -

war. Rotenone_has a low toxicity in mammals but its toXicity .

against.fish and a variety of inseCts,but'not all, 'is quite .

high. It has been said that the ideal insecticides are the

compounds which have both-the lethal action . of rotenone and

the paralytic action of pyrethroids. Therefore the pharma-

cological importance of the studies on rotenClids lies in the

action mechanismof totenone, the relationship between the

chmiCal structuree and physiological activities of totenone

derivatives, and the cause of the selectivity, of the toxicities

of rotnoidb.. .

As for the action mechanism of rotenone, blocking

of the activity of mitochondrial L-glutamic dehydrogenase

3,4) by rotenone was first pointed out , and later the

blockage was narrowed down to the NADH enzyme system. The

latest tudies proved that the blocking took place speci-

fically at the coupled oxidation of NADH and a ubiquinone**,

as shown in figure 1 577) .. The mechanism of the blocking at

this site of blockage was also studied using 14 -rotenone8) .

* Translator's Note: Derris elliptica, common in Malaya. tt ** or NADH 2 and a flavoprotein.

7 8

-

O .

Rote n (me. - Rote ri One: c(e h'freiVires

-

V D H N IDN de. b,rdrofe eur set Co Q Cyt Cyt

lk s-44..cce n . deltraroje mase

Fij. I: Si-tes of chi ri of kofeno ids ( crtYro )

Since the site of action.rotenonoidS is.probably

the same in fish, insects and Mammals, the selectiVity of

the rotenoids must be caused before the rotenoids reach, this

si te. The metabolism . of 14 -rotenone and-its selective C

toxicity against different kinds of living.crattires have

been studied by Fukami and others 9 ' 10) When 14 -rotenone

was .reacted with rat liver microspmes and NADPH as an

auXiliary enzyme in the -atmosphere, almost all the rotenone

was metabolized. Pive* major metabolic prodiicts which were

soluble in ether wereas shown in Figure 2,.hydroxylation

products at the isopropenyl side chain . and at the Junction

of B and C rings. When the same 1.4d-rotenone metabolism

was studied in vitro, .using liver Microsobes of mouse and

*Translator's Note: 4 in Figure 2. 8 in the preceding chart.

S L-CC C a. ci. ct

carp, abdominal microsomes of diazinone-,resistant houseflies,

. and those of the Wamon (or ring) . dockrbach'exactly the same

.hydroxylaticn products:as- found in the - rat livr'microsome

expriment wer detected,.and no qualitative difference

could be detectecLamong the metabolites. At the:same time,'

a series. of in vivo - experiments '1,,?ere - conducted by administering

14 0-rotenone to the various living'creatures aformntioned,

and the metabolism products were examined, by extracting

various organsof the aubstrate and their . urine samples. .

with ther. he ether soluble metabolites were found to be

cmpletely identical:hydroxylation products as found . by

'in vitro experiment. Furthermore; the biological activities.

'of . these hydroxylated products were much . weaker_than the .

starting material, rotenone,, and, therefore it was assumed

that the oxidative metabolisM of rotenOne is a type of .

detoxication.

8'-hydroxy 8'..-hydroxy 8'-hydroxy: rotenolone II rotenone rotnolone I

I rotenolone II 4-- rotenone -) rotenoldne I

1. I 6',7'2dihyclro- 6',7 1-dihy- droxy roxy rotenolone I rotenolone II rotenone

Metabolites of rotenone

10 -

C H30 OCH

I 3 roteV\0131ete. I Oh

rOtehOlOner. Oh

cH z.

d14 2. 0H CHz0H

OH 113

22 : 1'4 eta. boh's yn of Rote none tr/o-, in vitro)

Ilacaled 17).- them s cx.tor .

HO

8 I e

O H

0 o

P ky ct r-o), rote n one.

H r- 2 ( c H3 ch, hyaroxy rote n o he,

As will be discussed in the section on pyrethroids,

piperonyl butoxide 11) , egonol12Y 1 sesame oil 13) and other

methylenedioxyphnols increase the insecticidal activity

of rotenone, nad therefore these compounds are insecticidal

synergists. It is also known that piperonyl butoxide,

sulfoxide, MGK 264 amd SKF-525A inhibit in vitro hydroxylation

of rotenone via the microsome - NADPH - oxidase system, These

findings indicate that the oxidative degradation of rotenone

by the mdcrosomes plays a significantly important role in

understanding the metabolisM of:rotenone in biological systemslo)

When in vitro liver-microsome-MADPH systems including rotenone

are prepared using rat., mouse.and carp lVers, and to each

system, supernatant of the corresponding liver extract is

added, the degration. prOgress beyond 'the:aforementioned

hydrooxylation stages,.prodUcing water-soluble mtabolites

by further transformation of the ether-solubie hydroxylation

prOdUcts. This finding leads to a hypotheis that the se-

condary metabolism products of rotenone probably yield a

variety of their functional derivatives which . themselVes

aid the exCretion. and .degradation of rOtenome1 .0) .. The

author plane t explain this hypothesis in more'details,

in the section on selective tOxicity of rotenone, near th

end of thie article.

- 12 -

O CH (0 CHI C H2)z. o C2 Hs- i CH3

sesa.kne X CHa C HC

112 (C CH1 0)z Ce,i H9

pifret-ony/ at-oxiWe

I It ci Hs- o

c- c-ocitz n N 2 I 2. fis-CI Hz C H3

S tc-F

Tian 5 letto Noie : All the siruciu.re s are corrected by the Mule inhrY

2.1.2 Pyrethroids

Pyrethrin has remarkablY distinct advantages in that

it is fast-acting and that itsi toxicity for mammals is sur-

prisingly . low, but it has the unfavourable property of per-.

mitting rather quick recovery of victims. The mechanism of

'its insectiidal_ . actiOn has hot been clarified yet. Phama-

cological interest in pyrethroids can be summarized in their

rapid paralytic action, low toxicity against mammals, and the

mechanism of their action. .

r 13 -

The . activities..of pyrethroids apPar to te': caused-

by two.partiai . structural fragment,:one bing cyclopropane-

'carboxylic acid moiety which contains an 'unSaturated side.

chain and the'other, cyclopentanolne which is also :

functionalized by an unaaturated :alkyi sid:chin..It is

said -that,.. - if.a slight modification is made in either One '

of these, acid and alcohol of pyrethrOids,: theiractivitie

are often remarkablilowered. .It has been also Said .that,,

in the detoxicative metabolism of pyrethroids, hydrolySis

of the ester linkage plays the most important . role. However,

the experimental results.obtained by applying 14 . -pyrethrin14).9

C ). ' allethrin -15 , and 14 -pyrethrin-I and -cinerin-I

16) - to C .

tousefiies showed that the amounts -of ChrYsanthemic-acid

formed by hydrolysis' were qUite small, that three.of the

five majometabolip produts isolated contained the un-

changed moiety of chrysanthemic . acid.in th ester form, and

-thatthe.remaining two major'product also retained the ester .

. linkage. When the latter esters were hydrolyZed and the

Denig test commonly usd as a qualitative test for, chrYsan-

themic . acid.was . apPlied to the hydrolysis Irroducts, the .

'16) tests were positive . Thse results:undoubtedly show that

.the detoxicative metabolim of pyrethroids in insectsdoes

not include the hydrolysis of the ester linkage as its major

metabolism route. . -

14

It has been known for some years that addition of

sesame oil to pyrethroids greatly.increases their insecticidal

activities. The causative substance in sesame oil responsible

for the synergic effect was examined, and based on the re'sult

of this study, synthetic synergists such as piperonyl

butoxide and sulfoxide, both of which had a methylene-

dioxyphenyl ring, were discovered. these are serving a

practical purpose. On other synthetic synergist, sesamex

increases the insecticidal effect of pyrethrin-I against

houseflies nine times, and that of cinerin-I twelve times.

As already described in the section on rotenone, 1,3-

benzodioxole, SKF-525A and the related compounds, which

block the activity of miCrosome . OxidaSes, were also reported

to increase the effect'ofpyrethrin against.houseflies and

soldier-bugs.Thee findings of the.sYnergic effects of

various chemical compounds seem to indicate that the meta-

bolism path of pyrethroids in their detxication processes -

is . through the oxidation route rather than the. ester hydrO .-

lysis.

Recently Yamamoto and others19) isolated thirteen

metabolites from 14 0-allethrin and ten from 14 c-PYrethrin-I,

using the oxidase system obtained from abdominal microsomes

of houseflies. The main metabolites were the oxidation product

of the isobutenyl methyl group of the chrysanthemic acid

moiety . of the pyrethroids (Fig.3).

1 7 ,18)

1 t Cili C = CR

/ c /C:= 0

0

Correcte4 6x te-* to

CH3 H3

- 1 5 -

c'H3 CH3

C H /

C 1 *1y> c

11 II o

s ee.osom e -FiVADPH -I- Oz.

Chi 3>C HO 0 C

CH3 Ch13

/ H d CHC C / fr( ft

fog"-

o

f-1 CHz-c= CC C fretirolome

if (-films trans 'tram s )**** R C e / e Hz cinerol on e 4441-

ry re tA tin I Rr--->

her i n

Fij. 3 : ajor rnel&bolic route of CA1751Lilfileir) itte

tAe .-transkt.for

..The kn.es-7- -Adits no 5 ijrn flica nce A S 0 ) rela.+1'0-e Comfly ura.hi on of 11-.-1:44-lempt

trot.? I's 'not shown (2) Siet n dardel proC424u.r ', ores - i1 esoro el-km-aeon of

cycj pro pane ckv- bEKy 11 'c a c"fef. tielp R ate re cr-trs 4? 4

* Add eei -t-A e

- 16-

At the same time, it was found that the same methyl group

Of 14 -dimethrin and 14 -phthalthrin, which were analogues

of pyrethrin, were also oxidized to the corresponding

carboxyl group. Therefore it is currently assumed that

the metabolisms of all the pyrethroids lidth the chrysanthemic

acid moiety follow the oxidation path as described above.

This assumption is supported by the finding that some .

PYrethroids, namely pyrethrin-II and cinerin-II, which lack

the isobutenyl group, are not synergized by sesamex as

cinerin-I and pyrethrin-I are 20) .

As for the in vivo metaboliem of pyrethroids in

mammals, Miyamoto and others 21) reported on 14 -phthalthrin

(3,4,5,6-tetrahydrophthalimidomethyl chrysanthemate) orally

administered to rats.Phthalthrin was slowly absorbed in

the alimentary canal, and the absorbed material was rapidly

degraded. The main metabolite was 3-hydroxy-cyclohexane-

1.2-dicarboximide produced by hydroxylation of the primary

hydrolysis product.

. . 2.2 Organophosphates* ' ./388 . . .

Parathion appeared on the market at the same' time'-

as DDT did', after the lastwar, and replaced rotenone,.

*Translator's Note: The author uses the term organic-phosphorus- insecticides. The translator uses the term organophosphate as

a generic term to cover all the organic insecticides con-taining phosphorus regardless of the types, that is, phosphate, phosphonate, phosphorothionate, phosphorothiolate etc.

.1 7 . -

pyrethrin and nicotine. It showed an excellent insecticidal

effect as a contact chemical. However, since parathion and

other organophosphate insecticides are also highly toxic to

the higher mammals, much time had to be devoted to find

better-qualified, less toxic organophosphate insecticides.

As for their pharmaological studies, the areas examined

in more detail are the action mechanism of the organophos-

phates with relatively low toxicity, the cause of selective

toxicity against insects and mammals, and the mechanism of

resistance induced by the organophosphatef3 among some strains

of insects; these studies have been done quite actively as

a part of basic metabolism study of various living creatures.

Another characteristic point about the organo-phosphate

research to be mentioned here is that, in comparison with

the studies of other insec .ficides, weedkillers.and fungicides,

this was the area in which the application of radioisotope

techniques was carried out since the earliest date of them

all. Indeed it is not going too far to say that most of the

organophosphate action mechanisms were done using the radio-

isotope technique.

2.2.1 Excharige Reactions between S and O.

The main cause of the toxic aCtion of organophosphate

insecticides is believed to be the disruption of nervous

activity caused by inhibition of the function of cholin-

esteras.e. Usually, however, the thiono type insecticides

1

18

(P = S) . do not block the cholinesterase activity, .while .

the phosphate ester group (P 0), which can be produced

by in vivo oxidation, show a very strong blocking power.

Therefore, the phosphate ester group is usually considered

to be principally responsible for the acti .iity, and the

in vivo oxidation process is called the activation reaction22) .

The activation enzyme of parathit on to paraoxon is found in

rat liver or Wamon cockroach microsomes, as other drug-

oxidizing enzymes, and it needs NADPH,and oxygen for its

functioning as the activator 23) . The enzyme activity is

inhibited by antiresistant, sesamex, piperonyl butoxide,

sulfoxide, and MGIC-264 23) .

Rats which had been treated orally with dieldrin 24)

' or with chlorcyclizine, phenobarbital, SKP-52511 25) were

shown to have resistance against parathion. The reasen for

this rsistance was proven . to be the increased activity of .

the A-esterase, which was responsible for the hydrolysis

of paraoxon, in their livers and.serum 25) , and the presence

.of another new dtoxication enzyme vas also pointed out 26) .

Nakatsugawa and others 27) studied the metabolism of 35 - S parathion using the rat liver and housefly microsome-NADPH

oxidase system and found- a new type of detoxication reaction

which split the phosphate ester linkage, in addition to the

" . - *Translator's Note: . The author does not - differentiate between '' 'phpi5nate, phospnonate, phesphorothiolat, - and..phosphoramidate', : although:all of them -contain the P . ..group. , -. .

_

;4;

,- H

E-e-o

pare( th l'OPI

M e 1 bo A's m (1'

414A 1 . 0k1 4y in l icroSopme

- 19

known activation reaction of parathion to oxon (Fig. 4).

Nea1 28) also obtained the sanie result using 322-parathion.

Furthermore, it is now known that rats pretreated with

barbitals have increased activities in both microsomal

detoxication and activation reaction 25) . By these findings,

ecife-Atiorel., E-L 0 _ 0 0 e z rniavsome 27/.0 Amprtl

Oz

Et 0 > P -OH Ef 0 .

pat-04.0x

de

it is confirmed that both the esterase and the oxidase

participate in the lowering of parathion toxicity after

pretreatment, but which one of the two different enzymes

le the more critical for the lowering of the toxicity is

not yet known.

2.2.2 Oxidation of Sulfur

Generally speaking, the insecticides classified

as the penetrating compounds do not loose their activity

even after the compounds remain in the plant tissue for a

considerable period. During this period, the sulfur atoms

often undergo oxidations. One typical thioether, demeton

is a mixture of thiono-type and thiol-type compounds.

(C 2 H 0) P(S)0C H SC H thiono n-type demeto 5 2 2 4 2 5

(C 2H S O) 2P(0)3C 2114 3C 2H5 thiol-type demeton.

The thiono-type compound is oxidized to an oxon type compound,

while the thiol-type compounds tindergo in vivo oxidations to

sulfoxides and sulfones (Pig. 5)29).

- 22 -

Fukuto and his coworkers 3(431) examined tlie

metabolism of 32 -demeton in beans and cotton plants and

found that it was converted to oxidation products which

showed considerable cholinesterase blocking.activities,

and they later confirmed the same metabolites were formed

in mammals as well. The first stage metabolites were mainly

sulfoxides, which later were converted to sulfones but this

second stage metabolism was found to be much slower than the

first stage metabolism32) . The thioether groups of 32p-

disyston and 32 -thimet were also oxidized in the plant 29)s

On the other hand, the oxidases that produced sulfoxides

and sulfones were found in mammalian livers and Wamon

cockroach microsomes, and the experiments using 32p-thiometon

showed that they required NADPH and oxygen for their

functioning, and their activities were blocked by SKF-525A

and piperonyl butoxide 33)

(C 2HS O) 2 P (S) S CH2 0112 SC211 5 Dysyston

(C 2H S O) 2 P ,(S) S 011 2 S 2 211 5 I Thimet .

(CHS O) 2 P (5) S CH2 .CH 2 S 02115 ThioMeton

2.2.3 Hydroxylations of Alkyl Side Chains and N-dealkylations

Since 'the early stage of studies on organophosphates,

it has been .known'that schradan (octamethYlpyrophosphoramide)

is *converted to its N-hydroxyme.thyl derivative 34-6)' and

5Wtrerq5eleeMIMMIVe.""

-

lately a penetrating insecticide, bidri [3- (di-methoxy-

phosphinyloxy)- N,N-dimethyl-cis-crotonamide] was also shown

to umdergo the same type of metabolism. Two compounds,

N-hydroxymethyl bidrin and N-demethyl bidrin which is known

commonly as azodrin, were isolated from the metabolite '

mixture of 32 -bidrin in rats, insects and cotton plants37) .

MenZer38)' later isolated N-hydroxYlmethyl aZodrin and

. .N-demethyl azOdrin using . bidriii.labeled with 14 0 and 32p ,

, and estimated that N-demethyl compound wathe secondary

metabolite of the. N-hydroxymthyl derivative. It wag quite

.interesting to.know that all these - four compounds had -

significantly.high insecticidal activities against hoUse

.flies (Table 1). This metabolism path was conSidered to be

participated in by an oxidase system, and the insecticidal .

effect against houseflies was greatly increased by addition

39) of the',common synerest, sesamex .

Table 1: Anticholinesterase activities and toxicities of . Bidrin and its derivatives.

CH 30 o H

,CH3O e-ID-C=

" CH 3 . o

Bidrin

Bidrin N-hydroxymethyl Azodrin ,N-hydroxymethyl N-denietliy1

Bidrin Azotlrin . Azodrin

/C1I 3 CH3 ,CH2 .

'Cl-l3

CH 201-i . .

24 -

TOCP (tri-O-cresylphosphate) is a very wellknown

synergist for the organophosphates which contain carboxylic

acid ester groups, as found in malathion. TOCP itself,

however, does not have cholinesterase blocking, power, but

when it is activated after being metabolised in a mouse body

or by a slice of its liver, it becomes a strong inhibitor

41) of cholinesterase 40) Eto and hie coworkers isolated a

cyclic phosphate ester, M-1, from rates orally administered

with TOCP, and found that the cyclic ester M-1 had the

cholinesterase blocking power, and proposed the metabolic

path of TOCP as shown in Figure 6. It is apparnt that one

of the ring-substituted methyl group was oxidized by the

oxidase system in the liver microsome, and then the oxidation

product cyclizes, through hydrolysis and intramolecular

rearrangement of the phosphate group, to the final product.

The last stage is probably caused by the action of plasma

albumin. Based on the structure of this active compound,

several active analogues have been synthesized and some

are being used as an insecticide.

2.3 Carbamate Insecticides

Carbamate insecticides generally inhibit the

cholinesterase activity as do the organophosphates. Against

mammals, their toxicities are usually low, and they have

fairly wide selectivities against insecte. being active

against leafhoppers (such as green rice leafhopper)* ,

- : - 25

planthoppers (such as smaller brown planthopper, brown

planthopper)*, and aphids (such as corn leaf aphids, green

peach aphid)* but inactive against houseflies and cockroaches.

The cause of these selectivities has been explained as the

difference in detoxication mechanisms rather than the

difference in the cholinesterase blocking power. Although

the pharmacological intrests in the carbamate insecticides

have been found in the same area where they were found in

organophosphates, since they have a wlder margin of selectivi-

ties, more metabolism studies specific to each insect are

. desirable.

The metabolism of the carbamate insecticides varies

depending on the subjects of study, mammals, i'nsects or

plants, but it is classified into the following three major

areas; (1) hydrolysis of carbamate ester group, (2) oxidations

(hydroxylations of ring and ring substituents, N-demethylation,

and N-hydroxymethylation of corresponding N-methyl groups),

and (3) s complex formations such as glucuronide formation,

glycidation, sulfonation or glycosidatione when metabolized

in plants, of the hydroxyl grouppand.carboxyl groups produced.

by the .aforementioned processes.

Only 1-naphthol was the isolable metabolite of

14 -carbaryl which was a representative carbamate insecticide 42)

*Transl.'Note: Added by the translator as these were the ones found in Japan. . , .

C11 3 0 8

0P-0

C11 3

NADPI1,0,

1-.yd r.oxy:r. e TOCP (9000) .

O

0 P 0 -

o

plasma album%

C113 0

0 P- /

11- 1 : ( 1. 2 X 10' )

- 26 -

Fig. 6 : Metabolism of TOCP 'Figure in ( ) is /390 relative anticholinesterase activity]

TOCP

( 1 )

at the early stage of metabolism study. However,Dorough

4 and others 3) later isolated several compounds shown in

Figure 7 from the metabolite mixture of carbaryl which was

labeled with 14 at naphthyl,,carbonyl and -methyl after

treating with a liver-microsomal NADPH oxidase system:. They

also isolated 1-naphthol which was -the hydrolysis product

and its derivative using liver homogenate. Recently, in

addition to the metabolism requence shown in the figure,

0-dealkylation, hydroxylation of ring substituents, and

kti,o1

creme

OH

0 it

0 dSHMe wt icrosome ' P H

02_

vAecrosome

NADPH 02. je

o L 141-1cFl 1oji

Sle o It " oixM . octsli-Me_

erect* r kyetrot-y

Cae 60..ry OfKict

OC (Me

27 -

Litre r mecros omai oix.:culobi of

Carbe. ry I iv% s ech'c i'de e v% t- o)

-.28 -

sulfoxidation were reported using thirt y. three different,

methyl and dimethyl carbamates which were labeled with

44) . 14 0 . Among these newly found metabolites, some were

even stronger cholinesterase inhibitors than the original

44) carbamates . Although the activity of the oxidase which

transformed carbamates into metabolites was inhibited by

piperonyl butoxide 45) , the oxidase itself was found, by an

in vivo experiment, to be-synergistic* just as sesamex was45-48)

Four glucuronides (shown above.) and sulfate esters

of the oxidation and hydrolysis products:were found as the

products of in vivo metabolism of carbaryl. labeled with .

methyl-14 0., carbohyl-I4 0 and naphthy1140 in rats and : guinea

pigs49) . In th metabolism of this compound, since a

rl.atively large amount of th baterial was not hydrolyzed,

the metabolic pathways were mainly xidations and derivative

formations. Two compounds, 1-naphthyl methylimidocarbonate

0-glucuronide, which is the direct derivative, and 4 -

(methylcarbamoyloxy)-lnaphthyl glucuronide were the major

metabo 1 ites 49) . The same metabolites were'foilnd in experiMent

using pigs, mnkeys and sheep 5) . libWeVer,..in the case of

14 0-3,4-dichlorobenzyl N-methylcarbamate, its hydrolysis

was the major mtabolic'rute at the initial stage, which

was followed by oxidation'to yield the corresponding

carboxyluc acid, wilich was . isOlated, and.which,' in turn,'

*Transi. Note: synergistic to the action of the carbamate , itself?

O Gr

0 Cq rrOz., Or

-v1,041.4-6 le 4 e.

VIA 0%10 r vA 0:410tek

ro n e

- 29-

0 CI- ' dzrzl Me

was converted to and isolated as the amide of glycine,

namely, 3,4-dichlorohippuric acid 51) . Metabolism studies

of penetrating carbamate insecticide, bano1-14 0 (2-chloro-

4,5-xyly1 methylcarbamate), and carbaryl-14 c were conducted

using bean plants. The major metabolites were found to be

their N-methyl derivatives 52) 0r glycosides of their aromatic

53,54) ring hydroxylation products

Another penetrating carbamate furadan (2,2-dimethy1-

2,3-dihydrobenzofurany1-7N-methyl carbamate)'was studied

using cotton plants, corn plants, houseflies, larvae of

salt marsh caterpillar and mice, and its metabolic pathways

in different species of plants. insects and'animals were

compared using 14 c and 3H markers. As shown in Figure 8, the

major paths of the.metabolism were essentially the same. /391

Namely, furadan was first hydroxylated to 3-hydroxy furadan,

of which the hydroxyl group was difficult to react . to produce

a derivative, and then the large portion of 3-hydroxy furadan

was oxidized to 3-keto furadan, which was hydrolyzed to give

- 30 -

'Me. oxid ox:d .

O=C N Me.

o 0 N ctizoH

o 0=e ts1 Me

'Me Fi

Fikrad an

0 H

COM

4-or-ma-Won.

e+0,1 s -re .0+ e u-, vn cretti-Wre s

f' . ylkyvt- s eYS e c..4- s

. 0

}10.

'

c l CI

3,4-clich1o"ro- henzyl N-methyl carhamate

0

CI l

3,4-dichloro-' hippuric acid

- 31 -

the corresponding phenol. The phenol produced various /391

derivatives 54) .

As for the selectivity of carbamatesi a few probable

causes for it have been cleared mainly based on their meta- 1

bolism studies. Namely, the susceptibility of honeybees was

estimated to be due to their relatively low oxidase potency 55,56)

The carvamates with low toxicities against mammals generally

were also easily detoxucgted and quickly excreted. On the

other hand, highly toxic carbamates such as furadan and temik

D-methy1-2-(methylthio) propionaldehyde 0-(methyl carbamoyl)

oximej were found to yield metabolites which also had the

same type of toxicities, and therefore in these carbamates

the in vivo metabolism is more likely of a nature of

activation of toxicity rather than detoxication56 ' 57) .

2.4 Organochloro Insecticides

Organochloro insecticides which include DDT, MO,

cyclodiene compounds and others, generally have small acute

toXicities against mammals but their insecticidal activities

are quite strong, and their residual insecticidal activities

are also high. The machanism of th insecticidal activities

has not been established. Since this group of insecticides

has been used since very soon after the war, appearance of

a large number of resistant strains of insects has been

recorded. The radoiseope technique has been frequently

applied to the exploration of the mechanism of the r sistance

formation.

;-,:;1

V,, b1

Generally speaking, the organochloro insecticides are

stable and difficult to decompose even in plants, soil '

and water, and they remain, without decomposition, for a

long period. Consequently, the chain of food intake and

excretion cycles tends to result in their eventual cumulation'

in vertebrates at high concentrations. The isotope technique

is therefore being used in the study of the metabolism of

these chloro compounds in mammalians.

Three major types of reactions, namely dehydro-

chlorination, oxidation, and reductive dechlorination, are

known as the metabolic routes of DDT (Fig. 9). Among thes

pathways, the dehydrochlorination of DDT to DDE is the

critical factor to determine the DDT resistance of house-

flies 58,59) .

As for the oxidation pathway, kelthane from the

Kiiroshojo-fly * was the first isolated metabolite 6) . Later

kelthane or kelthane-like substances were isolated from the

housefly, Chyabane-cockroach ** , and Sashi-game***, when

labeled DDT was used 61,62) . The oxidases of-these insects

had the same characteristics as those of mammalian liver

microsomal oxidase, requiring NADPH and oxigen to oxidize ++61). DDT and their activities being accelerated by Mg 6l

Note: domestic fruit fly, Drosophila melanogaster.

" Literally brown-wing-cockroach. The reference (61) shows German cockroach.

*** II " Sashi-tortoise. It must be an insect.

- 33 -

t.

OH FJ

0:5Cl.? CL.C. CL

,of f kel 4-tuule.

H Ct. j>--C N

Cl

DT

b.(11/ 7# (p)Ci!- C

it

D D E ce>'d

rm'cr- osome , WAD I) Oz

ID P

eat) oks (Y.'s ot DDT

tcLt- f (4) - CLC., CL CLC CL

CL

kyietro4(.314*e

iDD T

Y (oc,L4- c - ? c ter oH

The enzyme activities were foUnd to be particularlY good

ing)arathion-resistant.houseflis 61) . The larvae of '

Sashi-gam were quite strongly DDT resistant, and the

, larvae treated with.SKF7525A increased the-effect of DDT,

and the yield of kelthane-like material in -their bodies

decreased. On the other:hand, 5-methylchlanthrene lowered

the effect of DDT", and the bodily content of the hydroxylated

.substance increased. Therefore.it is quite sound to

conclude that the MicrosOmal oxidative . mtabolism. vas ,

63) te cause of the DDTresistance ,

Chlordane, heptachlor, aldrin, isoaldrin, dieldrin

and endrin are all highly chlorinated hydrocarbons and they

are synthesized by diene condensations. The first four of

the afore-listed insecticides are epoxidized in the body,

and the epoxides are physiologically active and are chemically

stable compounds. Epoxidation is quite well-known in insecte,

in addition to the same found in mammals64 e 65) . Namely, the

formation of heptachlor epoxide from 14c-heptach1or66) , and

the epoxidation of aldrin to dieldrin and that of isodrin

to endrin 67) are some - of the-well-known examples. The

symptoms of poisoning by these chemical insecticide

in insects are parallel to the progressing of the epoxidat1ons 67) ,

and, in order to cause the poisoning symptoms supply of

oxygen is needed 67) . Sesamex, which is a common synergist,

decreases the activities of aldrin and heptachlor against

houseflies and this has been explained as due to the inhibition

of epoxidation of these chemical s68) . Ail these results con-

firmed by in vivo experiments clearly indicate that the

metabolism mechanisms of these are deeply related to the

reaction mechanism of microsomal oxidase.

Liver microsomal enzymes of rabbits and rate epoxidize

heptachlor 69) , aldrin69) and isodrin70) . and the enzymes

require NADPH and oxygen for the readtion and their potencies

53)* are lowered by SKF-525A, piperonyl butoxide and parathion

Tranel. Note: 58) ?

observed

CI

CI '011.

ilydroxychlordene

Cl

Cl

Chlordene epoxide

*Transl. Note: 74) cannot be found.

() 11

II

35

In the case of insect e , the epoxidations are reportedly

71) carried out by cockroach fat-body microsomes and the

71- ) 'housefly abdominal microsomes 73 /392

. Dieldrin which is the product of epoxidgtion of .

14 0 -aldrin waa further tranformed to highlY polar . trans-.

6,7 7dihydroxy7dihydro , aldrin (aldrin glycol) by.the expoxide-. 75-78)! ring opening reaction, in - insects and in mammals

(Fig. 10). The physiological actiVity of this compound

against insects is approximately. one twelfth. of that of

77): dieldrin ,. and the enzyme repponsible for the epoxide

opening was found in livr microsOmes of'housflies and

mamMais 79)

Fig. 10: Metabolisms of aldrin and dieldrin (in vivo, in vitro)

ci 1.1 . n o

to-

atNI a cl . cI Aldrin Die Idrin glycol

In the case of diene compounds, in addition to the

epoxidations by the microsomal oxidase system, hydroxylations

have been recently reported. For instance > 14 -chlordene

yields epoxy compound and hydroxychlordene which is not .

63) (or 68?) toxic

'

This hydroxylative detoxication of chlordene is

carried out by an enzyme whose functioning mechanism was 80) 'verified in part by in vivo synergistic effect by sesamex

2.5 Inductive Activation of Drug-oxidizing Enzymes bY

Organochloro Insecticides

Consecutive administration of barbituric acid to

mammals, and the resulting drug-resistancy have been studied

quite well. The mechanism of resistance to barbituric acid

was explained as the result of increased liver microsomal

81) capability to hydroxylate barbituric acid . If liver-

carcinogenic 3'-methy1-4-dimethylaminoazobenzene is

administered .together with 3-methylcholanthrene, the ' 82) former's carcinogenic actiVity is entirely suPpressed.

and this is now known as a result.of.ah increased activity

level of the micrOsomal oxidase, which . became capable of .

converting the carcinogen into non-carcinogenic substances. . . .

.The number of these componds, Which are able to increase.

the drug-metabOlism activity.of liVer microsomes, as shomin

by barbituric acid and 3-methylchlanthrene,: is increasing.

recently, akd this phenomenon ia called induction of drug-

metabolizing' enzymes by drugs, and.those. drugs which increase

the activities of enzymes are called.enzyme Inducers 83)

Intensified Potency of microsomal oxidase by insecticides

has also been found in numerous.cas. The*.first insecticide

84 .1 85) Shown to be an inducer was chlordane 1 and besides._14at,

.Chlordane .

Methoxychlor

DDT -;

DDE

DDD Kelthane

Endrin

Dieldrin

Aidrin

*Heptachlor

Heptachlor epoxide

BHC

37

various other chloro insecticides shown in Table 2 were

proven to be induce rs86) . The same effect of induction

was a],so confirmed in DDE, whicii was a non-toxic metabolite

of DDT.

Table 2: Organochloro insecticides that promote metabolism of drugs. .

As one 'of the in vivo functionings of the micro-

somal enzymes, its relation to steroid metabolism in

mammals has been clarified. Further, the relationship

between the steroidal metabolism and the drug-induced

activity of the oxydase system has been reported by conney 87)

He found that, elen phenobarbital was administered to rats,

their, liver microsomal potency to hydroxylate steroid rings

of testosterone and androstenedione increased drastically.

It is indeed exiting to find that the hydroxylations of

steroid hormones by liver microsomes can be subjected to

the same effect as the drug-metabolizing enzymes are. It

is therefore important to investigate how various drugs

that influence the activities drug-metabolizing enzymes

ultimately effect the general physiological system of bodies

through the changes of steroid hormone metabolism. '

- 38 -

The effect of organochloro insedticides on the

steroid metabolism is becomming somewhat clearer88) , and

currently it is estimated 89) that the rapid decrease of

the feathered tribe in Europe and in North America might

have been caused by the imbalance of pex hormones owing to

the chloro insecticides which generally show high residue

rates. For example, pigeons treated with either DDT or

dieldrin or both showed higher contents of polar metabolites

of 14 progesterone and 14 testosterone due to the increased /393

activities of their liver microsomeNADpH oxidase, and the

metabolites after treatment with DDT as well as with diel-

drin were found to be different (Fig. 11).

Fi. 11: Chromatography of Testosterone metabolites in pigeon liver as induced by chloroinsecticides.

-- DDT *-G- Die!chin

DDT+ Dieldr.IN Compel

distande'Of Moving (col) .

On the other hand, the livers of untreated control pigeons

did not indicate the presence of these highly polar

metaboiites. The results undoubtely suggest that there

- 39 -

. is always a possibility of drug-induced imbalance. of . sex

hormones, and this is the basis for the speculation 'of the

decrease,of birds'owing io their poor reproducton89) . As

quite a' lot of reports 'are. prePared regarding:the. '

induction of insecticides, more effort should be.devoted

to clearing the.possible inhibition of reproduction bY . various

other pesticides as well as their metabolites.

3. WEEDKILLERS

.In the sections on insecticides the author described- .

their metabolic patterns,forcussing hie attention on the

oxidative metabolism in mammals,.insects and plants, and

.discussed the width of the seleptivities of insecticides.

In the field of weedkillers, the - selective ssceptibilities'

'of plants to chemical substances and the caue. of the

selectivities are alsO most important subjects. As expected,'

we find a rather large number of examples of application Of

radioisotopes especially' in the studies of the absorption:

mechanism- of the plants, the . mobility Of chemicals in the

plants-, and their metabolic pathway. In addition to these

examples, since most weedkillers are applied.to the soil,

the stability, -Mobility and availability of weedkillers in

the . soil; their degradation in the soil, and their metabolism

re.soil bacteria are important problems. Therefore, we see

a considerable number of research reports in these areas as

well.

- 40 -

H "...N./H (Cill') (C3111)

H 1-4C3

3.1 Trifluralin

Trifluralin(040t-trifluoro-2,6-dinitro-N,N-

dinormalpropyl-p-toluidine) is a toluidine-type weedkiller,

and is commonly used to destroy weeds of the rice-plant

family and other common wide-leaf weeds. The degradation

of'14 -labeled trifluralin is different depending.on whether

the conditions are aerbic or unaerobic..NaMely aerobically,

dealkyIation'is the first Step of.degradation in the soil,

which is then follOwed.by reduction of the nitro group, but

reduction before dealkylation is the first step of anaerobic

) clegradation9 . The plants which show resistance . agaihst this

.0,N NO, , 0,N f'. 1 NO3 03N e).1 NH,

CF, Trifturalin

CF, .CF, 6.q.7

decdkyi I no e ci eri ect ve

chemical were found to be poor absorbers of the chemical

from the soil90) , and the major metabolities in carrots

were found to be the dealjyl compounds 91)

3.2 Di phe nami de

This acid amide type weedkiller has high mobility

in sOil, and is an excellent weedkiller for weeds of the

rice-plant family and annual wide-leaf weeds. The Metabolism

studY Using a 14 c-labeled marker in plants shoWed that

- 41 -

demethyl compound (N-methy1-2,2-diphenyl acetamide) was

. the major metabolite, and the yield of thi conversion

96)*' was extremely high . On the other hand, when the same

. marker was administered to rats, N-demethyl compoune),

N-hydroxymethyl compound(s) and their derivatives, 0- and

N-glucuronides, and 0-sulfate were isolated from the urine,

indicating that the transformation of the N-methyl group s

was the major metabolic pathway and the other possible route,

that is, hydroxylation in the aromatic ring, was almost

negligible.

Diplienzu.nid

3.3 Diuron and Monuron

Both diuron D-(3,4 7 dichloropheny1)1,1dimethyl

urea3 and'monuron D r-(p-chloropheny1)-1,1-diMethyl urea]

re phenyl urea-type weedkillers: They are absorbed by the

roots and accumulated in the leaVes and inbibit the Plants'

photosynthesis. All the weedkillers which have the -NH-00-

group, namely,'carbamate-type, urea-type, triaZine-type and

anilide-type weed-killers, block the Photosynthesis, and

it.is assumed that the' hydrogen bridge formation between

*Translator's Note: 92)-95) are in section 3.8

this atomic group of the weedkillers and the free imino

group, hydroxyl group and the carbonyl group of the

chlorophyl-protein complex in the plant leaves is the cause

of clocking photosynthesis.

Resistance and susceptibility of a few plants against

diuron and monuron were examined using radioisotopes. There

was no difference in the mode of absorption or moving and

distribution in the plants between the cotton-plant, which

was relatively resistant to these chemicals, and the bean

plant, which was susceptible to theme Although both mono-

demethyl derivative.and di-demethyl derivative, which* were

toxic to plants, were commonly isolated as the metabolites

(of diuron) **' *** , the toxic demethyi derivative(s) could

not be found in the metabolites from the cotton . plant.

Similar demethyl derivative(s) were isolated as the metabolites

of monuron. Therefore, the selectivity difference between

these plants against these chemicals was considered due to

the quantitative difference in detoxicative metabolism 97) .

Recently it was reported that an oxidase in cotton leaves

oxidatively removed the methyl group(s) of 14 -monuron to

produce N-demethyl derivative(s) 98) . This enzyme was found

in microsome fraction as the sametype of enzyme did in

mammals and insects, and it required NADPH and oxygen to

*Transl. Note: either one or both? ** " tt added by the translator

from which plants?

do the job. This was : the -first : .exaMpl of cxidation.of

pesticides by a plant oxidase and therefore it

noteworthy.

0 CI ,_=,) C2N a "i5 < CH' 11 e C711,

011, N.-- Cit

Neron

o ' r Nm7.!:

- 44 -

99) susceptible or resistant . When 14 -dicamba Was administered

to rats, most , of the drug was excreted in the mine, and about

1/5 of it was a glucuronide drivative and the other major

portion was unchanged dicambalW)

C001-1

. M crrlba.

3,5 Paraquat, Diquat

Both paraquat (1,1=dimethy1-4,4=bipyridinum salt)

and diquat (6,7-ehydrodipyrido pyrazinum

salt) appear to block the photosynthesis of plants. Since

they are quickly deactivated in soil, they are usually applied.

directly on leaved and stems. Both 14 0-paraquat and diquat

are relatively stable in plants, and are only difficultly

metabolized 101) , but they are quite susceptible to soil-

microbial decomposition. Microbes first demethylate paraquat

to 1-methy1-4,4' -dipyridinium ion in which the two htero-

aromatic rings are oxidatively cleared to yield 1-methyl-

4-carboxypyridinium ion. This sequence is entirely different

from the photodegradative reaction, in which the formation

of 1-methy1-4-carboxypyridinium takes'place (immediately)*

after the first step of the degradation, i.e. splitting of

the hetero ring (instead of de-N-methylation) .

*Transl. Note: added by the translator.

1

* 10 2 , 1 0 3 )

1 -methyl -4 -carboxy-: pyridinium ion '

3.6 Simazine, Atrazine

+ -

(C11 3 - br)CNr C11 3 CI 13 N1-

. .

Paraquat 1 -methyl- 4,4'- dipyridinium ion

-4

Triazine-type weedkillers cannot'sterilize - the

Weedss : seed,' and their reation mechanism is estimated

to,be identical With that-of .urea-type naMely,

they are absorbed by the rots and cumulate in the leaves,

where theublock the plants' photosynthesis by. hydrogen:bond

formation with the chlOrophyl-protein.cOmplex that.is

critically important for the:photosynthesis. Bth simazine

C,2-chloro 74,6-bis(ethylimino)-s-triazinelandiatrazine

C2-chlor6-4-thylimino-6-isopropylmino .-s-triaiine3 have

strikingly slective actractives against ,corn plantS and

'againbt weeds. The metabolism of simazine was examined by

applying labeled simazine to the. resistant.corn plant, and

it was learned that the detoxication by the plant was mainly ,

caused by the plant's ability to substitute the chlorine atom

with a hydroxyl group. This non-harmful hydroxy simazine

was actually synthesized non-enzymatically by a reaction .

of benzoxazinone (2,4-dihydroxy-3-keto-7-methoxy-1,4-

benzoxazine) which was found in the plant. The concentration

of this latter compound in a plant and the drug resistance

CI

C

C3 H8SH-C C- NHC,H . N o

Simazine OH

Ces - NH-C-C-NHC11 ,,

>se- .

1-Iydroxy Si 'mazine

a

N/S.,..N

(C1:13)MCON-C u4114C,I1, N O

Atrazine

ci

II C;HeNII-C,...,; .A1.. NH.

Simazineffixl-Jkft:YdeethY1 derivative

- 46 -

of

the plant was repOrted tobe very closely.corrlate 104-106).

However, there has been tarepoi-'t(s) t -o debate the relation-

.

Another metabolic route of triazine chemicals in

plants is dealkylation. Shimabukuro studied the metabolism

of 14 -atrazine in bean, and found 2-chloro-4-amino-6- C

isopropylamino-s-triazine, which was.the principal metabolite,

but no hydroxy triazine derivative 107,108). This deethyl

compound was also active in the inhibition of photo-

synthesis although its activity was lower than that of

atrazine itself. Bean plants are not as strongly resistant

/395 to atrazine as corn plants are, but are more strongly

resistant than oat. Therefore it was estimated that.the

plants which were moderately resistant to triazine-type

weedkillers might be detoxicating atrazine by deethylating

some portion of the absorbed atrazine 108). It was also

confirmed that the triazine-type weedkillers were hydro-

10)* xylated in sciil 7 , and a soil microbe, Aspergillus

* Transl.Note: Misprint of 109)?

3.7 Propanil

- 47 -

fumigatus converted 14 0 -

simazine to 2-ch1oro-4-amino-6-

ethylamino - s-triazine by deethylation as bean dis ilo)

An anilide, propanil (3i4-dichloroproPion'anilide)

is a contact-type weedkiller, and it seIeCtively destroys.

annual weeds such as barnyard'grass, mehishiba* and other

weeds of the rice-plant family,. lthYough it hardly.damagee.

'rice:plants. The mechanis .mHof the 'selectivity of this weed

killer was studied in.detail and the results shoWed that

the differnt activities against'rice.plant.and barnyard

gras.s were not caused by the.difference in absorption and ,

- mobility in these two plants but:by:the degree of the

detxication reaction,.which wasthe hydrolyis of.propanil-

.111 to 3,4-dichloroaniline (DCA)and propionic aci -114)d .

It was fOund that. rice plant at its third-leafing.,age .

had 26 times as large a detoxication activity as that of

barnyard grass113) . The enzyme which decomposes propanil

in rice plants was studied by Freer and others115) . It is

also known that the organophosphates and carbamate insecti-

cides'blocked the activity of this enzyme, and this finding

can explain why the mixture spraying of this weedkiller

and organophosphates or carbamate insecticides caused the

116)115 112 ill effect on the sprayed rice plants '

.* Transi. Note: water-chestnut?'

15 1-I-C-CH .(0111C11., - I

NH C-C,113

48

' CI - r"c y".CI Cl CI CI

Propanil DCA DLA . . . .

Recently. Yih and otherS 117) proposed . a different

Mechanism'.of prOpanil metabolism after analyzing the -

metabolites obtained. from carbonyl and ring labeled propanil.

TheY suggested that the first step of the degrdative meta-'

bolism was the oxidation to.3,4-dichlorolactanilide '(DLA)

which was then hydrolyzed to.DCA and lactio - acid rther . than

to propionic acid, While the older mechanism assumed th

direct hydrolysis of the anilide linkage to yield DCA . and

proPionic.aCid.

The fata of 14 -43CA prodUced in rice:plants after

degradation of propanil has been the .subjectof many studies.

Sti i 1 118) discoVered four DCA derivatives, one-of which. was

N-. (3,4-dighloropheny1)-gluccisamine. ih 119)* clarified that

other.DCA , derivatives were a mixture ofiDCA - glycosides-of

glucose, xylose and fructose and other unidentified DCA

glycosides. However,.the .content of these Water-soluble

DCAcarbohydrte coMpounds was rather small and a major

portion of the DCA derivativesSfas .the complex compounds -

of DCA and lignin cellulose, hemicellulose and other high

->Trnsl. Note.: addect.by the translator.,

molecular weight cll comPonent. The change Of the side

chain propionat of 'propanil was eXamined Using a:marker .

qabeled at its terminal methyl group and at the Carbonyl.

.It was Confirmed . that a large portion of the propionate ,

group became 14 2 before being absorbed. by the plants. CO'

This was explained by the rapid . hydrolysis of the propionate

amide group.to propionic acid, which underwent P-oxidation t

produce CO2 120)

Since propanil decomposes quickly in soil and is

deactivated, the weeding effect by the soil-treatment method

was said to be very poor. This rapid dcomposition was found

to be due to the hydrolysis of propanil to DCA by soil

bacteria121,122). Acylamidase which exists in mammalian

livers was also known to decompose propanil l23).

3.8 Naphthaleneacetic Acid

The wellknOwn plant hrmone substance, 1-naphthalene-

acetic acid is metabolized to itsderivatives in plants. Th

major metabolites isolated were aspartic acid . amid

EN-(1-naphthalene acetY1) aspartic acid] and "a glucoside

(0-naphthalene actyl glucose). An oxidation product,

8-hydroxy-l-haphthalene acetic acid and its. glucoside . were

also identified92 ' 95) .'The distributions of these metabolites.

were examined using 14 0-market compound, but they were quite

different depending on the plants examined94) , On the other

hand, most of the labeled compound was excreted in urine

DCooH it I C ii

C .00H

H2 CO 0 H

I v)alelni-kale me,

l lAkco s ct e, 31-y c e ae-ritrcx-Wve,

szx.s rem-11 % c c . 4 ctg r mkt' (re.

ti

CC-Hz 0 im.e09'1 CH.4HC.CO0H, 0*

when administered to rats, and the glycine derivative

(naphthaceturic acid) and the corresponding glucuronide'

95) were the major metabolites .

4. FUNGICIDES

The number of .examples of application of radio-

isotopes in the field of biochemistry of flIngicide was

relatively small, in comparison to thosein insecticides

and weedkillers.-The reaSon for this is found in the facts

that most of the reaction mechanism - of the.fungicides Was

solved without using radioisotopes, that, although in'the

application of insectiClidesi numerous.problems Such as ,

comparative toxiciV:studies between higher-vertebrates

and insects, selective toxicities among the insects, and

the mechanism of resistance, and also in the field of weed-

killers, the problems of selective toxicities among plants

were solved mainly with radioisotope techniques, the same

types of studies were not very important when application

of fungicides was studied, and that actually most of these

problems were solved without the aid of the radioisotope

technique. However, in recent years, a large number of new

fungicides have been developed and are being used or about

to be used. Naturally more knowledge about the mechanisms

of the action of fungibides, of metabolism and resistance

of fungicides and further data on the metabolism in higher

plants and animals are needed, and the studies using isotopes

4) are rapidly increasing. Since Kuwasaki 12 already reviewed

the general application of radioisotopes in the studies of

fungicides, this author discusses only those newer fungi-

cides, namely, organochloro fungicides and organophosphates,

reported after Kuwasaki's review..

PCBA (Pentachlorobenzyl alcohol) is,a well-known

fungicide to protect rice plants from rice withering disease.

It does not have fungicidal activity against the fungi -

responsible for the disease when they are placed in a test

tube or on rice plant leaves but it prevents rdce plants

from contracting the disease. However, only little was

known about the 'mechanism of its functioning. In order to

clum cil

ci ci ci

ci -Vji ci ci ' ciVci

CI . , ci CI

PC IA PentacIdire- Pentachloro-

benzylatdekde . benzoic acid .s%

O0 11 .

52.-

clarify the action mechanism 'or the toxicity of PCBA, its

metabolisms in fungi, plants and animals were studied.

When rice plants were treated with' 14 c-PCBA, there was

little radioactivity in the aqueous extract, but the ether-

soluble part contained unchanged PCBA and two unidentified

125) . . 126) metabolites . Kakinoki and other detected pentachloro-

benzaldehyde and pentachlorobenzoic acid from rice plants

treated with PCBA, ,and they estimated that oxidations of

the side chain were the major metabolic route- On the other

hand, when 14 0-PCBA was orally administered to rats, only a

small amount of the chemical was absorbed and found in blood,

liver and urine, but the major portion was excreted in the

feces with no change in its structure. The metabolites

found in the urine were pentachlorobenzoic acid and PCBA

glucuronide 127) .

In order to clarify the action mechanism of a similar

organochloro fungicide, PCPA (pentachlorophenyl acetate),

absorption and transformation of the14 c-labeled fungicide

in the pathogenic fungi of rice withering disease and in

rice plants were studied. In both fungi and plants, the

absorbed PCPA was hydrolyzed to pentachlorophenol (PCP).

Therefre the fungicidal - actiVity of PCPA was probably

cased by ita metabdIic PCP128) cased'by ita metabdIic PCP

EDDP (0-ethyl S,S-diphenyl phosphorodithiolate)

is an organophosphate type fungicide and it shows either

curative or preventive effect on rice withering disease. The

mechanism of action has not yet been clarified. Uesugi 129)

reported on its metabolism in the pathogenic fungus of the

disease. 1%me1y 32p-EDDP was quickly.absorbed into the fungi

and hydrolyzed to inorganic phosphoric acid via 0-ethyl

S-phenyl thiophosphoric acid and ethyl phosphoric acid.

At the initial stage of this metabolic pathway, an inter-

mediate metabolite, which was soluble in toluene and was

fungicidally active, was detected. There was no difference

in the metabolic patterns of EDDP by susceptible fungua and

resistant fungus.

As to its metabolism in plants 130) , was rapidly

subjected to hydrolysis, and its initial metabolites were

0-ethy1 S-phenyl thiophosphoric acid (dephenyl derivative)

and S,S-diphenyl dithiophosphoric acid (deethyl derivative)

and later as the metabolism progressed, 0-ethyl phosphoric

acid ana phosphoric acid increased in the'metaibolite mixture.

o

7 e.

dphenyl derivative

0 sO

.EDDP

'deethyl derivative

-- 54 -

It is reasonable to assume that the degradation of EDDP

in rice plants took place in the order of the intermediate

compounds listed above.

1) Fukami and others13 compared the metabolism

pattern of 32p-EDDP in various microbes and animals, namely,

Bacillus subtilus, Fusarium, rice withering disease fungus,

cockroach and rat. The patterns Of metabolites found in

water-soluble and chloroform-soluble fractions obtained

from each species were nearlY identical. The 32p-EDDP Orally

administered . to rats was almost exclusively excreted in the

urine, nd watrsoluble metaboiite in the rats' tissue and

urine were the cam as'found in rice . plants. In the 'case of

,cogkroaches, the results were "again the same...SeruM,.liyer

microsome, supernatant Of liver and other tissues of rats

contained enzymes whiCh'hydrolyze EDDP,'and these enzymes .

appeared to participat.in ihe metabolism .of EDDP. Un th

other hand, body tissues of rats and cOckroaches, and their.

exCrements Contained some in viv: .metabolites which .Wer

soluble in chlorciforM and which were not the nyrolysis

_

7?

- 55 -

products described above. Since these unidentified metabolites

appearedto Widentical with the oxidative metabolites of

EDDP with the NADPH-oxidase system obtained from the.liver

and fat-body microsomes of rats and cockroaches, they'were

considered to be ring hydroxylation products. Further these

substances were identified from the metabolites of rice

withering disease fungi, after treatment with EDDP, although

they were not found in the metabolites of EDDP-treated

Fusarium and Bacillus microbes. They are currently estimated

to be the same toluene-soluble materials isolated by Uesugi

and others, as described above.

IBP (0,0-diisopropy1 S-behzyl phosphorothiolate)

is also an effective fungicide against rice withering

disease and it is an organophosphate type chemical. Its

metabolism in the pathogenic fungi of rice withering disease

was examined using a 35 s and 32p doubly labeled IBP. IBP

was taken - in very rapidly, and its major portion was hydro-

lyzed to 0,0-diisopropyl thiophosphoric acid. As the

metabolites other than the hydrolysis product(s), two

toluene-soluble intermediate metabolites were found. There

was no difference in the patterns of metabolism between

the IBP-Susceptible and IBP resistant-fungi l .

0 . 3>

CI-10) e-S-CH;Ct I-1, 0H3 t .

.

IBP , .

L. ,

56: -

'

5. SELECTIVE TOXICITY-

In order to clarify the cause of the . seledtive

toxicity of pesticides, it is important to examine every

step , of the interactions between the pesticides and the

substrates, plants or animals, until their pharmacological

activities'are revealed by the symptoms of the substrates,

and how the difference of the chemical structures of the

pesticides and the difference of the substrates are reflected

in the interaction of the chemical and the substrate must

be analyzed at each step, physicb-chemically and biochemically.

As factors in the selectivities of chemicals may be counted

the absorption of the chemical, its mobility , and distribution

in the body, its routes of metabolism and the mechanism, and

further the sites of action of the chemicals. In this review,

the author so far has explained the width of selectivity

of insecticides, weedkillers and fungicides individually.

In the following sections, the author attempts to describe

the selectivities of a few insecticides, of which the

principal cause of the seledtive pharmacological action

was undoubtedly shown to be either oxidation or metabolism

through complex formations.

5.1 Seiective Toxicity of Rotenone

Rotenone is' a selective insecticide. Its site 's of

action are in the electron transfer system in the respiration

process of insecte as are its sites of action in mammals.

57

As already described, the mtabolites of 14 0-rotenone by

the microsomal system of mammals or insects are almost

exclusively hydroxylated compounds, and the metabolites

formed were essentially the same regardless of the sub- -, 10) . 0) estima strates' - ' 9; Fukami and others 1 9 ' ted that

there were three factors participating in the selectivity

of rotenone against insects and mammals.

The first factor is the quantitative difference of

detoxicative activities by oxidations. 14 c-Rotenone is

oxidized stepwise to various hydroxylated derivatives

which are less toxic than rotenone, by microsomes of various

living creatures. his activity of detoxication is the

strongest in rat liver; the cockroach fat-body has a weaker

activity than the aforementioned and the middle intestine

of cockroaches has an even' weaker activity (Table 3).

Comparison of the contents of P-450, which is a component

of microsomal oxidase systems, reveals that, in cockroach

fat-body, it is less than 1/7 of that in rat liver microsomes.

This low relative content alone indicates that the activitY

of detoxicative metabolism of rotenone in cockroach must

be very low'.

Ether layer 4 1 c (%'

15 13 ' 31

'cockroach fat body

1 1

cockroach middle intestine 63 24, 10

61 . 24.

rat liver 63 15 .

cockroach middle intestine

1 1.

- 58 -

. . . . .. , . . . . . .. .Table-3:- letabolism of . rotnone'by cOmbined.micrdsomes . . .

Or microSoMe sUPernatants Of.inseCts or 'animais . .

Combination

Mcrosome suiDernatant rotenone rotenone hyroxylated . mtabolites

Aqueous layer 0 '(%). .

rat liver

rat liver rat liver 76

cockroach fat body

68 .16

18

-59

The second factor is sthe difference in the activities

of secondary reactions by supernatant enzymes of various

tissues and organs of the insecte and animals. Although the

hydroxylated metabolites of rotenone have only low pharma-

cological activities, they are still toxic. These metabolites,

however, can be converted to either insoluble, non-toxic

metabolites, when supernatant fractions of liver are added.

The newly derived metabolites are considered to be a complex

of unknown structure. The activity of the supernatants to

promote the secondary reaction varies considerably depending

on the source of the supernatants, and the supernatant from

cockroaches can hardly complete the secondary reaction,

while that from rut liver rapidly finishes the reaction.

The third factor is the effect of the-natural: .

, inhibitor, which exists in insect body' tissues, on the

oxidative . metabolism. The-sUpernatantsof.cockroach fat-body

and middle intestine contain. protein-like natural inhibitor

which depresses the oxidative metaboliem: Ifthe . supernatants

. of the aforedescribed'fat-body or middle intestine are .

'added, the activities of microsomal oxidase systems from.

. rat liver and cockroach fat-body, and the yields of the'

hydroylated Metabolites are considerably lowered. Particularly

the middle intestine supernatant hass quite strong inhibitory

activity. On the other hand, eupernatants from* mammals do

not show this type of activity (Table 3). Even though a lot

of work has to be done on the physiological effects of the

natural inhibitor on insect metabolism, it is pr.esently

estimated that the inhibitor is suppressing the detoxication

of rotenone in the bodies of insects by oxidation.

Since the patterns of in vitro enzymatic metabolisms

by rats and cockrOaches resemble very well those of in vivo /398

metabolism observed in orally administered rats or in

cockroaches injected with rotenone, the former in vitro

patterns are interpreted as exact replicas of the metabolism

lof rotenone in each insect or animal. Accordingly, the

selectivity of rotenone has the width of the marginal

selectivities both in the in vivo microsomal, primary

oxidative metabolism and in the portion where the secondary

* reaction participated 10) .

5.2 Selective Toxicity of Parathion-Type Insecticides

Dialkyl phosphate type insecticides have lower

toxicity against mammals when the alkyl group are methyls

than when they are ethyls. However; against insects, both

have the same insecticidal activities with no selectivity.

Plapp and other 133') labeled the methyl ar ethyl phosphate

ester with 32 ' and examined their metabolisms in mammals P

and in insects. They found that, in mammals, the methyl

ester produced non-toxic demethyl compounds as a product

*T.ransl. Note: . ?

()stalky/ pkos 'hs..ip cm.c,t,des

pa ratil l 'o 11,

rte. fh o PI

e_. E

of P-O-Methyl splitting, but the ethyl ester did.not yield

deethyl deriVative or, if yielded, only a very little

amount. On the other hand, in insects, dealkylative

metabolism appeared to take place only very difficulty.

4- . Fukami and others13 6) independently fond that there was

an enzyme which demethylated methyl ester insecticides, in

liver supernatant of mammals, using 32 2-markers. This

enzyme acted only at the methyl ester group, and the activity

of that from the mammalian source was high but that of the

insects was quite low. These results agreed well with

Plapp's results described above. Thus the difference in

dealkylations, that is, demethyiation of methyl ester

insecticides to yieid non-toxic deriVative and deethylation

which is difficult to take place, or the difference in the

activities of enzymes that caused the demethylation,

probably the principal cause of the low, toxicity against

mammals and no selectivity among insects.

0)P - O -L,t4

OLe me tivy parectkeon

GrS- 1LF CH

vnee I tk

y a g

I Del ra.d. 04.+%lo o fvlefky l tet r a. till' 0 v% 11Ak+txtk t' one s- le-M y I -t- ra.s -t-exc&se_

Aecti-ki N

yaxathor,. ey

-4 Tree. s I o.+ar s Weete * 0 be coYrec.t , e1-te r c4wet&1 or MOV10 yn e rot.ra.-t-h

I rar VI

The demethylation enzyme requites - reduced glutathione

for the reaction. This indicates that the demethylation is

not a simple hydrolysis of the phosphate ester, but a

transfer reaction of the methyl group from the phosphate

ester to glutathione. Experiments using'a 14 0-Methyl marker 157)

proved that.the'transfer was assisted.ty a kind:of

glutathione-transferase. .

C}130>

AluFb P-0 NO z

(7. e. )

- Sumithion

Sumithion, - which.i also a methyl phosphate insecticide

with only lightly.different Chemical structure .from that -.

of methylparathion, ha only 1/36 the toxipIty of that of .

methylparathion. 'This selectivity was aIso studied using . .

various markers". .First, it was found that th e degradeion

velocities of-methylparathion and - sumithion bY the afor-.-

described demethylase were nearly identical, and, the possible

eelebtivity owing to this enzyme was de1,1ied136,138)...' Other

possible . Participating enzymes Were sOught after but none

of . the results uld eXplain,the.selectivitY 138) . Next, -

Miyamoto and others 139-14,2 ) administered sumithiOn,'methyl-

parathion and their oxons to Mammalei and examined their .

activations in 'thebodies, their-degradations, degrees of. ,

inhibition of cholinesterase actiVities, and mobilities.in .

the tissues. They found that sumioxOn .penetrated mUch more

slowly into brains than methylparaoxon, and the animals'

brain-nerves were not ttacked seriously, and concluded

that the,difference in the Mobilities into the brain is

the main cause of the selectivities. However, Hollingworth

and others 143) discovered that, when a large dose of

sumithion was given to mice, almost all of the sumithion

Ut

-64

degraded in the bodies, and about 80% of the degradation

products was demethyl derivative, and they, therefore,

proposed that the demethylation was the critical factor

in their selectivities, thus casting doubts on Miyamoto

and. others' brain- blodvessel. theory.

The cause of the low toxicity of sumithion is still

a subject of serious debate, and perhaps no one single factor

la the critically important, low toxicity cause, and a

number of factors may be contributing their influences

simultaneously. It might be too optimistic to give any

conclusive explanation of this problem, but the author .

favours the following hypothesis the difference in the

toxicities for animals between sumithion and methylparathion

lies in the difference in the Velocities of the insecticides

in reaching the brains. Owing to the slow mobility of

sumioxon, before the brain nerves are paralyzed, both

sumithion and sumioxon are rapidly detoxicated by the action

ofdemethylase. The low toxicity of sumithion must be a .

summary effect of the two actions.

I C.>- '11

Bibliography in jaDanese

33) Fukami, J. and others: Abstracts of Annual Meeting (1968), APplied Zoology and Entomology (Odokon-Koen) (1968) (page missing)

83) Kato, R.: Sites of Action of Drugs (Yakubutsu no Sayoten) (Ed. by Takagi, H.) p. 227, Nankodo

Pub. Co., Tokyo (1968) ' 112) Adachi, M. and others: 15roduction Technology of

Pesticides (Noyakusisangijutsu) 14,19 (1966); 11 (1966).

114) Ishizuka, K. and others: Abstracts of Annual Meeting of Japanese Agricultural Chemical Society p. 249 (Nichinoka Koen Yoshi) (1967).

124) Kuwazuka, S. : Radioisotopes, 16, 37 (1967).

125) Ishida, M.: Abstracts of Japan - U.S.A. Pesticide Seminar (Nichibei Noxaku Seminar Koen Yoshi) 189 (1967)

126) Kakinoki, K. and others: Abstracts of Meeting of Japan Plant Pathology Society (Nippon Shyokubutsu Byori Gakkai Koen Yoshi) 34 (1968).

127) Ishida, M. and others: Abstracts of Annual Meeting of Japanese Agricultural Chemical Society p. 48 (Nichinoka Koen Yoshi) (1969).

128) Nishiki, 111. and others: Abstracts of Meeting of Japan Plant Pathology Society (Nippon Shyokubutsu Byori Gakkailoen Yoshi) 189 (1968); Abstracts of Annual Meeting of Japanese Agricultural Chemical Society, p. 250 (Nichinoka Koen Yoshi) (1968). .

129) Uesugi, Y. and others: Abstracts of Meeting of Japan Plant Pathology Society (Nippon Shyokubutsu Byori Gakkai Koen Yoshi) 372 (1968).

130) Tanken, S. (or Tan, K.) and others: Abstracts of Annual Meeting of Japanese Agricultural Chemical Society, p. 149 (Nichinoka Koen Yoshi) (19 69).

131) Fukami, J.: Abstracts of Annual Meeting of Japanese Agricultural Chemical Society, p. 149 (Nichinoka Koen Yoshi) (1969).

132) Tomisawa, O. and others: Abstracts of Mee