Translation Series No. 1503

FISHERIES RESEARCH BOARD OF CANADA Translation Series No. 1503

Toxicological and biochemical research of pesticides using radioisotopes.

By Junichi Fukami

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

• From: • Hoshàseibushiteu (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 Fe-e /5 o3

DEPARTMENT OF THE SECRETARY OF STATE SECRÉTARIAT D'ÉTAT • .. TRANSLATION BUREAU BUREAU DES TRADUCTIONS FOREIGN LANGUAGES DIVISION DES LgANGUES DIVISION , CANADA ÉTRANGÈRES

TRANSLATED FROM - TRADUCTION DE INTO - EN Japanese. English

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 - RÉFéRENCE EN ANGLAIS Radioisotopes

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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

, f / 5-0 DEPARTMENT OF THE SECRETARY OF STATE SECRÉTARIAT D'ÉTAT ' eTRANSLATION BUREAU BUREAU DES 'TRADUCTIONS FOREIGN LANGUAGES DIVISION DIVISION DES LANGUES ÉTRANGÈRES

CANADA

CLIENT'S NO. DEPARTMENT ' OIVISION/BRANCH cm, ' •N9 OU CLIENT • MINISTERE DIVISION/DIRECTION VILLE 769,- 1 8-14 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

Review Article /385

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 . corrécted. 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 REVISÉE .nforrnzCien soule.ment

SOS-200-10-31

CONTENTS . . ...... . . . Page (Original • 1 • introduction . . • • - 2 • . (385)

.2- Insecticides - *. ". ' • 7 (386) • . • 7 . 0 .. 2.1 Insecticides ' . .. . • ' 2:1.1 Rotencids . . • 7 • 2.1.2 Pyrethroids- . 12 . (387) 2.2 OrganôphOsphates 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 ., .22 2.3 Carbamate Insecticides • ., 24 2.4 Organochloro Insecticides • .. 31 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

II • 4 Fungicides • 50 5 Selective TOxicity 56 (397) 5.1 Selective Tocicity of Rotenone 56 5.2 Selective Toxicity of Parathion Type Insecticides 60 (398) Bibliography • 65

• .40.

2 -

I. INTRODUCTION

The remarkable advances in developing various pesticides 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 agricultural 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** arè.organic-chemically synthesized compoundè. 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 théy'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 Yàesner 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 developéd. They suggested that (a)*the•use 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-: çides, 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 . generà1, 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:thèselasic 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 - fàr mammals and-destrby only the harmful insects. The différence in the'activities of these insecticides - against insecté and, maffimals is mostly depending.on . the qualitative and quantitative differenCes of the metabolism systems of theâe living'creatures. The•Metaboiisms of the-pesticides'and other chemicals are, .

mainly, depending on the action of their ' enz ymes. 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 peàticides. The firàt step • in the metabolism'of the pesticides introduced into the body ' is'probablY oxidation„reduction and hydrolysis, and the second àtep is the formation.of complexes of their primary . .metabOlism products. The procesées - 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 studieè of the areas not directly related to the pharmacology of insects.

As for thé oxidation of the Chemicals, it:is•Well • . 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 ià the system which includes - .c.itoChrome P-450.. The chemical•subàtances, once introduced . into the biological systàms, 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 pésticides•are . introduced - lnto biologiàal 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 pestiàidès 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 microsomes 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 mechanism•of totenone, the relationship between the • chémiCal structuree and physiological activities of totenone derivatives, and the cause of the selectivity, of the toxicities of roténoidb.. .

As for the action mechanism of rotenone, blocking of the activity of mitochondrial L-glutamic dehydrogenase by rotenone was first pointed out 3,4) , 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 8) this site of blockage was also studied using 14 -rotenone .

* 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 LÀ-CC C a. ci. ct 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 C -rotenone and-its selective toxicity against different kinds of living.créattires have 9 10) been studied by Fukami and others ' 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 were„as 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. 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 livér'microsome expériment weré 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 aforéméntioned, and the metabolism products were examined, by extracting various organsof the aubstrateè and their . urine samples. . with èther. he ether soluble metabolites were found to be • cômpletely 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 roténolone I I rotenolone II 4-- rotenone -) rotenoldne I I 6',7'21.dihyclro- 6',7 1-dihydroxy roxy rotenolone I rotenolone II rotenone

Metabolites of rotenone — 10 -

OCH roteV\0131ete. I Oh C H30 I 3

à rOtehOlOner. Oh

O o 0 H P ky ct r-o«), rote n one. cH z. d14 2. 0H - 2 ( H r CHz0H c H3 ch, hyaroxy rote n o he, OH 113

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

Ilacaled 17).- them s cx.tor . HO

8 I e • As will be discussed in the section on pyrethroids, piperonyl butoxide 11) , egonol 12Y 1 sesame oil 13)and other methylenedioxyphénols 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 systems lo) When in vitro liver-microsome-MADPH systems including rotenone are prepared using rat., mouse.and carp lïVers, and to each system,• supernatant of the corresponding liver extract is added, the degration. prOgresséé beyond 'the:aforementioned hydrooxylation stages,.prodUcing water-soluble métabolites by further transformation of the ether-solubie hydroxylation prOdUcts. This finding leads to a hypotheèis that the secondary 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 tà explain this hypothesis in more'details, in the section on selective tOxicity of rotenone, near thé end of thie article. • - 12 -

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

CH3 sesa.kne X CHa C HC 112 û (C CH1 0)z Ce,i H9

pifret-ony/ at-oxiWe

o I It ci Hs- c- c-ocitz n 2 N 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 insectiçidal_ . 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 of their action. . mechanism r 13 -

The . activities..of pyrethroids apPéar to te': caused- by two.partiai. structural fragment,:one béing à cyclopropane- 'carboxylic acid moiety which contains an 'unSaturated side. chain and the'other, cyclopentanolône which is also : functionalized by an unaaturated : alkyi sidé:chàin..It is said -that,.. - if.a slight modification is made in either One ' of these, acid and alcohol of pyrethrOids,: theiractivitieà 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, 14).9 the experimental results.obtained by applying 14 C. -pyrethrin -15 ). ' 16) - to allethrin , and 14 C -pyrethrin-I. and -cinerin-I • tousefiies showed that the amounts -of ChrYsanthemic-acid formed by hydrolysis' were qUite small, that three.of the five majometabolip produôts isolated contained the unchanged moiety of chrysanthemic . acid.in thé ester form, and -thatthe.remaining two major'producté also retained the ester .

. linkage. When the latter esters were hydrolyZed and the • Denigé test commonly uséd as a qualitative test for, chrYsan- themic . acid.was . apPlied to the hydrolysis Irroducts, the . '16) tests were positive • . Thèse results:undoubtedly show that .the detoxicative metabolièm 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„1 7 ,18) to increase the effect'ofpyrethrin against.houseflies and soldier-bugs.Theée findings of the.sYnergic effects of various chemical compounds seem to indicate that the metabolism path of pyrethroids in their detôxication processes - is . through the oxidation route rather than the. ester hydrO-. lysis.

Recently Yamamoto and others 19) 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 5 -

c'H3 CH3 1 t C H Cili C = C—R CH3 / / C c /C:= 0 H3 1 *1y> c • — 0 11 oII s ee.osom e -FiVADPH -I- Oz. CH3 ■Ch 31 • d Chi / H 3>C CH—C —C / •• fr( ft HO 0 C fog"-

f-1 ry re tA tin I Rr---> CHz-c= CC C fretirolome if (-films trans — 'tram s )**** her i n R —C e / e Hz cinerol on e 4441-

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

Correcte4 6x tAe .-transkt.for• ••

te-* to• ..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

cycjà 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 pharmaéological studies, the areas examined in more detail are the action mechanism of the organophosphates 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 radioisotope 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 parathiton 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) ' 25) with chlorcyclizine, phenobarbital, SKP-52511 or were • shown to have resistance against parathion. The reasen for • this résistance 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 détoxication enzyme vas also pointed out 26) . 27) Nakatsugawa and others 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 • '' _ 'phôpi5nate, phospnonate, phesphorothiolaté, - and..phosphoramidate', : although:all of them -contain the P . ..group. , -. .

;4; - 19

known activation reaction of parathion to oxon (Fig. 4). 28) Nea1 also obtained the sanie result using 32 2-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 rniavsome — 0 e z 27/.0 Amprtl £,- û H Oz pat-04.0x° E-e-o

pare( th l'OPI Et 0 > P -OH Ef 0 . de

M e 1 bo A's m 414A 1. 0k1 • 4y in licroSopme

(1'

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.

n • (C 2 H5 0)2 P(S)0C2 H4 SC2 5H thiono -type demeto

(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 metabolism 32) . The thioether groups of 32p- disyston and 32 -thimet were also oxidized in the plant s 29) 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 011 2 SC2115 Dysyston

(C 2H S O) 2 P ,(S) S 011 2 S 2 2115 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, bidrià [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 plants 37) . MenZer38)' later isolated N-hydroxYlmethyl aZodrin and . .N-demethyl azOdrin using . bidriii.labeled with 14 0 and 32p , , and estimated that N-demethyl compound waéthe secondary metabolite of the. N-hydroxyméthyl 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 of the',common synerest, sesamex 39) .

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

• • CH30 o • H • >e-ID-C=è ,CH3O

N-hydroxymethyl Azodrin ,N-hydroxymethyl N-denietliy1 Bidrin Bidrin Azotlrin . Azodrin • /C1I3 CH3 ,CH2 . CH201-i • . . ••• •

-N \, -N<' -N< • -N 'Cl-l3 CH:OH • H. H I t . -- 01}0 7.2 7.0 6.8 6.9 • • 6.5 hoi-isefly CliE lq.: ',D A in • 1.0 drop%ve cufa-n. 'housefly 38 14 . 6.4 30 . LI )1.0 mg/kg 3 . 14 . 18 • 8 12 Ç4.9.ryllilai i.kiIL 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 of cholinesterase 40) Eto and hie coworkers 41) 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 apparént 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 intàrests in the carbamate insecticides have been found in the same area where they were found in organophosphates, since they have a wlder margin of selectivities, 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. . , • . - 26 -

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

C113 0 TOCP 8 ( 1 ) 0—P-0

C113

NADPI1,0,

O 1-.yd r.oxy:r. e 0— P— 0 - TOCP (9000) . o

plasma album% C113 0 1■1- 1 0— P- : ( 1. 2 X 10' ) / •

at the early stage of metabolism study. However,Dorough and others 4 3) later isolated •several compounds shown in Figure 7 from the metabolite mixture of carbaryl which was labeled with 14 at naphthyl,,carbonyl and É-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 — 27 -

vAecrosome o L 141-1cFl1oji NADPH 02. je Sle o oixM . octsli-Me_It " erect* r kyetrot-y kti,o1 Cae 60..ry OfKict • 0 wt icrosome it OC (Me °creme ' P H 0 dSHMe 02_

Litre r mecros omai oix.:cuÀ‘lobi 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 14 0 44) . Among. these newly found metabolites, some were even stronger cholinesterase inhibitors than the original carbamates 44) . 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 naphthy114 0 in rats and : guinea pigs 49) . In thé metabolism of this compound, since a• large amount of thé baterial was not hydrolyzed, rèl.atively the metabolic pathways were mainly àxidations 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 experiMenté using pigs, mànkeys and sheep 5°) . libWeVer,..in the case of 14 0 -3,4-dichlorobenzyl N-methylcarbamate, its hydrolysis • was the major mètabolic'rôute 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? - 29-

0 CI- ' O Gr dzrzèl Me

0 Cq • VIA 0%10 r vA 0:410tek «rrOz., Or

ro n e -v1,041.4-6 le 4 e.

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) 0 r glycosides of their aromatic ring hydroxylation products 53,54)

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 3 H 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 o ox:d . 0=e ts1 Me O=C N Me. 'Me Fi Fikrad an o 0 N ctizoH

• • 0 H COM 4-or-ma-Won.

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

f' . ylkyvt- s eY‘S e c..4- s

. 0 0 • • Ç}10.

cl CI CI él 3,4-clich1o"ro- 3,4-dichloro-' henzyl N-methyl carhamate 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 • ;-,:;1 V,,b1

• 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 56 57) activation of toxicity rather than detoxication ' .

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 thé 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 ré sistance formation. 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 dehydrochlorination, oxidation, and reductive dechlorination, are known as the metabolic routes of DDT (Fig. 9). Among thesé pathways, the dehydrochlorination of DDT to DDE is the critical factor to determine the DDT resistance of houseflies 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 DDT and their activities being accelerated by Mg ++61).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 -

OH ‘7# (p)Ci!‘- C FJ b.(11/ it 0:5Cl.è?—ç H D D E CL.C. CL • Ct.‘ j>--C N t. ,of ce>“'d f kel 4-tuule. rm'crosome , Cl WAD I) Oz Y DT tcLèt- fè (4) (oc,L4- c - à? c ter oH - CLC., CL • CLC CL CL

ID P kyietro4(.314*e iDD T eat) oks (Y.'s ot DDT

The enzyme activities were foUnd to be particularlY good 61) ing)arathion-resistant.housefliés . The larvae of '

Sashi-gamè 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 . métabolism. 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 observed 67) in insects are parallel to the progressing of the epoxidat1ons , 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 confirmed 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 are lowered by SKF-525A, piperonyl butoxide and parathion 53)*

Tranel. Note: 58) ? • — 35 —

In the case of insect e , the epoxidations are reportedly carried out by cockroach fat-body microsomes 71) and the 71- ) 'housefly abdominal microsomes 73 /392

. • Dieldrin which is the product of epoxidgtion of .

14 0 -aldrin. waa further tranèformed to highlY polar . trans- 6,7 7dihydroxy7 dihydro, aldrin (aldrin glycol) by.the expoxide-. ring opening reaction, in - insects and in mammals 75-78)! (Fig. 10). •The physiological actiVity of this compound against insects is approximately. one twelfth. of that of dieldrin 77):,. and the enzyme repponsible for the epoxide opening was found in livèr microsOmes of'houséflies and mamMais 79) • Fig. 10: Metabolisms of aldrin and dieldrin (in vivo, in vitro) • • ci 1.1 • . () 11 n o to- atNI • a II • 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 . toxic 63) (or 68?) CI Cl

CI '011. Cl ilydroxychlordene Chlordene epoxide

*Transl. Note: 74) cannot be found.

' This hydroxylative detoxication of chlordene is carried out by an enzyme whose functioning mechanism was 'verified in part by in vivo synergistic effect by sesamex 80)

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 capability to hydroxylate barbituric acid 81) . If liver- carcinogenic 3'-methy1-4-dimethylaminoazobenzene is administered .together with 3-methylcholanthrene, the ' former's carcinogenic actiVity is entirely suPpressed.82) 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 compoùnds, Which are able to increase. the drug-metabOlism activity.of liVer microsomes, as shomin by barbituric acid and 3-methylchôlanthrene,: 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.caàés. The*.first insecticide Shown to be an inducer was chlordane• • 84 .1 85) 1 and besides._14at, — 37 • — various other chloro• insecticides shown in Table 2 were proven to be induce r s 86) . 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. .

.Chlordane . DDD Aidrin Methoxychlor Kelthane *Heptachlor DDT • -; Endrin Heptachlor epoxide DDE Dieldrin BHC

• As one 'of the in vivo functionings of the microsomal 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. ' - 3 8 -

The effect of organochloro insedticides on the 88) steroid metabolism is becomming somewhat clearer , 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 microsome—NADpH oxidase, and the metabolites after treatment with DDT as well as with dieldrin 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 89) . As decrease,of birds'owing io their poor reproducton 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 süsceptibilities' 'of plants to chemical substances and the cauée. 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 pathwayà. 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 -

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 aeràbic 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

• (Cill') (C•3111) 1-4C3 H H "...N./H

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

CF, • CF, ‘.CF, Trifturalin 6.q.7

decdkyi « I no e ci eri ect ve

chemical were found to be poor absorbers of the chemical from the soil 90) , 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 thié conversion was extremely high 96)*' . 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 the major metabolic pathway and the other possible route, was 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] àre 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 : .exaMplé of cxidation.of pesticides by a plant oxidase and therefore it was particularly noteworthy.

0 e •C711, CI •,_=,)› 11 C2N a ■" i5 • •

• Neron Monuron o ' r Nm7.!:<, • `11 • .

- .-Monuron monodemehyl derivativb:

3.4 Dicamba

• . picamba. 2-Methoxy-3,6-diChlorobenzoic adid dOeS not show any activity against GraminaCeas plants but is an. excellent weedkiller- for wide-ieaf'vffleds. It is said that, among its analogues, the 9nes with no substituent atthe • Para position ofthe benzoic carboxyl group are more active. The selective activity as - described above is lost if the

. methoxy grouP is substituted with Clorine.:Metabolites of dicamba were examined by applying labeled dicamber to dicamba7resistant wheat-and Zuzumeno-katabiral`. When the . metabolite mixture waa-hydrolyzed.with glucosidase or an acid, 5-hydroxy-2-methoxy-3,6-dichlorobenzoic acid Was • - isolated as the principal metabolite, in addition to a. .smaller amount of the - O-demethyl derivative,.3,6-dichloro- salicylic acid. The kinds of metabolites.isolated and their relative ratio were identical regai'dless of the plants, *Transl.Note: literally sparrow's-mourning-formal - 44 -

99) • susceptible or resistant . When 14 -dicamba Was administered 1 to rats, most , of the drug was excreted in the mine, and about 1/5 of it was a glucuronide dérivative and the other major portion was unchanged dicamba lW)

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 hétero- 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 * 10 2 the hetero ring (instead of de-N-methylation) . , 1 0 3 )

*Transl. Note: added by the translator. -4

■ . . + • - (C113 - br)—CNr C11 3 CI 13 ï N1- •Paraquat 1 -methyl- 4,4'- dipyridinium ion

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

3.6 Simazine, Atrazine

Triazine-type weedkillers cannot'sterilize - the •Weedss : seedè,' and their reaçtion mechanism is estimated to,be identical With that-of .urea-type naMely, they are absorbed by the roôts and cumulate in the leaves, where theàublock the plants' photosynthesis by. hydrogen:bond formation with the chlOrophyl-protein.cOmplex that.is critically important for the:photosynthesis. Bàth simazine C,2-chloro 7 4,6-bis(ethylimino)-s-triazinelandiatrazine • C2-chlor6-4-èthylimino-6-isopropylémino.-s-triaiine3 have strikingly sélective 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

•

• • • • • • - 46 -

of the plant was repOrted tobe very closely.corrélate à104-106). However, there has been tarepoi-'t(s) t-o debate the relation-

• .

CI • a

C • N /S.,..N • •• • C3 H8 SH-C C- NHC,H (C1:13)MCON-C u4114C,I1, . ■N o N O■ Simazine Atrazine OH ci

1 Ces - NH-C-C-NHC11,, II >se- . -C,...,; .A C;HeNII .»—. NH. 1-Iydroxy Simazine' Simazineffixl-Jkft:YdeethY1 derivative

Another metabolic route of triazine chemicals in •plants is dealkylation. Shimabukuro studied the metabolism

bean, and found 2-chloro-4-amino-6- of 14C -atrazine in 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 photosynthesis although its activity was lower than that of

atrazine itself. Bean plants are not as strongly resistant

to atrazine as corn plants are, but are more strongly /395 resistant than oat. Therefore it was estimated that.the •

plants which were moderately resistant to triazine-type

weedkillers might be detoxicating atrazine by deethylating 108). some portion of the absorbed atrazine It was also confirmed that the triazine-type weedkillers were hydro- 10)*7 xylated in sciil , and a soil microbe, Aspergillus

* Transl.Note: Misprint of 109)? - 47 -

fumigatus converted 14 0 simazine to 2-ch1oro-4-amino-6- • - ilo) ethylamino - s - triazine by deethylation as bean dis

• 3.7 Propanil

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,. àlthYough it hardly.damagee. 'rice:plants. The mechanis mHof. the 'selectivity of this weed killer was studied in.detail and the results shoWed that the differènt 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 • detàxication reaction,.which wasthe hydrolyÉis of.propanil- .111 to 3,4-dichloroaniline (DCA)and propionic aci d . -114) It was fOund that. à rice plant at its third-leafing.,age . had 26 times as large a detoxication activity as that of barnyard grass 113) . The enzyme which decomposes propanil 115) in rice plants was studied by Freer and others . It is also known that the organophosphates and carbamate insecticides'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 ill effect on the sprayed rice plants 112 115 ' 116)

.* Transi. Note: water-chestnut?' — 48 —

I • NH C-C,113 151-I-C-CH.(0111C11., - •

' 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 degràdative meta-' bolism was the oxidation to.3,4-dichlorolactanilide '(DLA) which was then hydrolyzed to.DCA and lactio - acid ràther . than to propionic acid, While the older mechanism assumed thé 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 .subject•of many studies. Sti i 1 118) discoVered four DCA derivatives, one-of which. was 119)* N-. (3,4-dighloropheny1)-gluccisamine. ïih 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 DCAcarbohydràte coMpounds was rather small and a major portion of the DCA derivativesSfas .the complex compounds - of DCA and lignin cellulose, hemicellulose and other high

->Trànsl. Note.: addect.by the translator., molecular weight céll comPonent. The change Of the side chain propionatè 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 CO'2 before being absorbed. by the plants. This was explained by the rapid . hydrolysis of the propionate amide group.to propionic acid, which underwent P-oxidation t • 120) produce CO2

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 décomposition was found to be due to the hydrolysis of propanil to DCA by soil bacteria 121,122). Acylamidase which exists in mammalian livers was also known to decompose propanil l23).

3.8 Naphthaleneacetic Acid

The wellknOwn plant hàrmone substance, 1-naphthaleneacetic acid is metabolized to its•derivatives in plants. Thé major metabolites isolated were aspartic acid . amidè EN-(1-naphthalene acetY1) aspartic acid] and "a glucoside (0-naphthalene acétyl 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 H2 CO 0 H C çii C è.00H

I — v)alelni-kale me, szx.s rem-11% c c . 4 ctg r mkt' (re.

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

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

when administered to rats, and the glycine derivative (naphthaceturic acid) and the corresponding glucuronide' were the major metabolites 95) .

4. FUNGICIDES

The number of .examples of application of radioisotopes in the field of biochemistry of flIngicideè 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 weedkillers, 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 are rapidly increasing. Since Kuwasaki 12 4) already• reviewed the general application of radioisotopes in the studies of fungicides, this author discusses only those newer fungicides, 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

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 metabolites 125) . Kakinoki. . and other 126) 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) .

clum cilô ÇO011 . ci ci • ci ci -Vjçi ciô ci ' ciVci CI. «, ci CI PC IA PentacIdire-• Pentachloro- benzylatdekde . benzoic acid .s%

In order to clarify the action mechanism of a similar organochloro fungicide, PCPA (pentachlorophenyl acetate), absorption and transformation of the•14 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). Therefôre the fungicidal- actiVity of PCPA was probably caùsed by ita metabdIic PCP 128) caùsed'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 intermediate 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. -- 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.

Fukami and others13 1) compared the metabolism pattern of 32 p-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

0 sO o

.EDDP 7 e. déphenyl 'deethyl derivative derivative

from each species were nearlY identical. The 32p-EDDP Orally administered . to rats was almost exclusively excreted in the urine, ànd watérsoluble metaboiite in the rats' tissue and urine were the camé 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 participaté.in ihe metabolism .of EDDP. Un thé other hand, body tissues of rats and cOckroaches, and their. exCrements Contained some in vivà: .metabolites which .Weré soluble in chlorciforM and which were not the nyrolysis

_ - 55 - products described above. Since these unidentified metabolites appeared•to 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 hydrolyzed 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;CtI-1, 0H3 t . • • L., . • IBP , . •

7?•• 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 métabolites 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) . 1 0) estima strates' - ' 9; Fukami and others 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. ïhis 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'. - 58 -

. .. , . . . . . . . . .. .Table-3:- •letabolism of . roténone'by. . cOmbined.micrdsomes . . . Or microSoMe sUPernatants Of.inseCts• or 'animais

Combination Ether layer 1 4 c (%'

Mïcrosome suiDernatant rotenone rotenone hyâroxylated Aqueous layer . métabolites . 0 '(%).

•rat liver

rat liver rat liver 76

cockroach fat body 15 • • 13 ' 31

1 1 'cockroach fat body 68 .16

cockroach middle intestine 63» 24, 10

1 1. cockroach middle intestine 61 . • 24.

rat liver 63 • 15 . •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 pharmacological 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.à protein-like natural inhibitor which depresses the oxidative metaboliem: If•the . supernatants . or middle intestine are • . of the aforedescribed'fat-body '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 32P ' and examined their metabolisms in mammals and in insects. They found that, in mammals, the methyl ester produced non-toxic demethyl compounds as a product

*T.ransl. Note: . ? ()stalky/ pkos ï'hs..ip cm.c,t,des

e_. pa ratil l 'o 11,

E rte. fh o PI

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. Fukami and others13 4- 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.

§Aecti-ki 0)P - O -L,t4 N OLe me tivy parectkeon GrS- 1LFCH ey yaxathor,. vnee y I atk 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_

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

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) a kind:of • proved that.the'transfer was assisted.ty glutathione-transferase. . • C}130 AluFb > P-0 NOz

(7. e. ›) - Sumithion

Sumithion, - which.i also a methyl phosphate insecticide with only àlightly.different Chemical structure .from that -. of methylparathion, ha é 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 aforé-.- •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 çàuld eXplain,the.selectivitY 138) . Next, - Miyamoto and others 139-14,2 ) administered sumithiOn,'methylparathion and their oxons to Mammalei and examined their . • Ut 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 — - 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- blôodvessel. 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.>-'11à

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