Sulfur in Pesticide Action and Metabolism 9780841206359, 9780841208100, 0-8412-0635-X

Table of contents :
Title Page......Page 1
Half Title Page......Page 3
Copyright......Page 4
ACS Symposium Series......Page 5
FOREWORD......Page 6
PdftkEmptyString......Page 0
PREFACE......Page 7
1 Low-Molecular-Weight Organosulfur Compounds in Nature: The Search for New Pesticides......Page 9
Acyclic Sulfur Systems......Page 10
Heterocyclic Sulfur Systems......Page 14
Conclusion......Page 16
Literature Cited......Page 18
2 Chemical Mechanisms of the Cytochrome P-450 Monooxygenase-Catalyzed Metabolism of Phosphorothionate Triesters......Page 23
LITERATURE CITED......Page 37
3 Sulfur in Propesticide Action......Page 39
Derivatized Carbamates......Page 40
Aryl- and Alkylsulfenyl Methylcarbamates......Page 41
Ν,Ν'-Thiodicarbamates......Page 43
Dialkylaminosulfenylmethylcarbamates......Page 47
N-Sulfinylmethylcarbamates......Page 48
(Alkoxycarbonyl) (alkylamino)sulfenylphosphoramidothioates......Page 50
Literature Cited......Page 52
Alkylthiotriazines and Thiocarbamates......Page 54
In vitro studies on the S-oxygenating enzyme involved in cyanatryn metabolism......Page 60
Further Reactions of the S-oxides......Page 61
Literature Cited......Page 64
5 Toxicological Significance of Oxidation and Rearrangement Reactions of S-Chloroallyl Thio- and Dithiocarbamate Herbicides......Page 66
Thiocarbamate Sulfoxides......Page 67
Dithiocarbamate Sulfines......Page 72
Metabolism of Thio- and Dithiocarbamates......Page 75
Toxicological Significance of Oxidation and Rearrangement Reactions......Page 76
Summary......Page 81
Literature Cited......Page 82
6 Biological and Chemical Behavior of Perhalogenmethylmercapto Fungicides: Metabolism and in Vitro Reactions of Dichlofluanid in Comparison with Captan......Page 84
Metabolism of (fluorodichloro 14C-methyl)Dichlofluanid in Strawberries......Page 87
Metabolism of (trichloro- 14C-methyl) Captan in Spinach and Soil......Page 90
In Vitro Studies......Page 91
Mutagenicity Studies......Page 93
Literature Cited......Page 95
7 Comparative Metabolism of Dithiolane Insecticides in Plants, Animals, and the Environment......Page 96
Metabolism in Rats......Page 97
Metabolism in Plants......Page 100
Photodegradation Studies......Page 102
Metabolism in Fish in the Rice Paddy Environment......Page 105
Literature Cited......Page 108
Properties of Iron-sulfur Proteins Low Molecular Weight, Terminal Iron-Sulfur Proteins......Page 109
Iron-Sulfur Proteins as an Electron Transfer Component to Cytochrome Ρ-450......Page 111
Involvement of Iron-sulfur Proteins in Pesticide Degradation......Page 113
Metabolic Degradation of Toxaphene by Pseudomonas putida......Page 115
Effects of Added Cofactors on Toxaphene Metabolism by Washed Cells......Page 118
Degradation of Mexacarbate in Cell-free Extracts of Bacillus megaterium......Page 122
Literature Cited......Page 125
9 Catabolism of Glutathione Conjugates of Pesticides in Higher Plants......Page 128
Methods......Page 130
Results and Discussion......Page 131
Summary and Conclusions......Page 152
Glossary of Chemical Abbreviations......Page 156
Abstract......Page 157
Literature Cited......Page 158
10 Role of Gut Microflora in Metabolism of Glutathione Conjugates of Xenobiotics......Page 160
Catabolism Utilizing Microfloral C-S Lyases......Page 162
Catabolism Utilizing Tissue C-S Lyases And Microfloral S-Glucuronidases In Addition To The Microfloral C-S Lyase......Page 169
LITERATURE CITED......Page 171
C......Page 174
D......Page 176
G......Page 177
M......Page 178
P......Page 181
R......Page 183
T......Page 184
X......Page 185

Citation preview

Sulfur in Pesticide Action and Metabolism

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

Sulfur in Pesticide Action and Metabolism Joseph D. Rosen, EDITOR Cook College, Rutgers University

Philip S. Magee, EDITOR Chevron Chemical Company

Joh University of California, Berkeley

Based on a symposium sponsored by the Division of Pesticide Chemistry at the Second Chemical Congress of the North American Continent, Las Vegas, Nevada, August 25-29, 1980.

158

ACS SYMPOSIUM SERIES

AMERICAN WASHINGTON,

CHEMICAL D.

SOCIETY C.

1981

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

Library of CongressCIPData Sulfur in pesticide action and (ACS symposium series, Includes bibliographical references and index. Contents: Low-molecular-weight organosulfur com­ pounds in nature / Eric Block—Chemical mechanisms of the cytochrome P-450 monooxygenase-catalyzed metabolism of phosphorothionate triesters / R. A . Neal —Sulfur in propesticide action / T. R. Fukuto and Μ. A . H . Fahmy—[etc.] 1. Organosulphur compounds—Physiological effect —Congresses. 2. Pesticides—Physiological effect—Con­ gresses. 3. Organosulphur compounds—Metabolism— Congresses. 4. Pesticides—Metabolism—Congresses. I. Rosen, Joseph D., 1935. II. Magee, Philip, 1926. III. Casida, John E., 1929. IV. Series: ACS symposium series; 158. SB952.S94S93 632'.95 81-7916 ISBN 0-8412-0635-X AACR2 ACSMC8 158 1-192

Copyright © 1981 American Chemical Society A l l Rights Reserved. The appearance of the code at the bottom of the first page of each article in this volume indicates the copyright owner's consent that reprographic copies of the article may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating new collective work, for resale, or for information storage and retrieval systems. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, repro­ duce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. PRINTED IN THE UNITED

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OF

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American Chemical Society Library In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.;

ACS Symposium Series; American Chemical 1155 16th St. N.20036 W.Society: Washington, DC, 1981. Washington, D. C.

ACS Symposium Series M. Joa

Advisory Board David L. Allara

James P. Lodge

Kenneth B. Bischoff

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Donald D. Dollberg

Leon Petrakis

Robert E. Feeney

Theodore Provder

Jack Halpern

F. Sherwood Rowland

Brian M . Harney

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W. Jeffrey Howe

Davis L. Temple, Jr.

James D. Idol, Jr.

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In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

FOREWORD The ACS SYMPOSIU a medium for publishing symposia quickly in book form. The format of the Series parallels that of the continuing ADVANCES IN CHEMISTRY SERIES except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. Papers are reviewed under the supervision of the Editors with the assistance of the Series Advisory Board and are selected to maintain the integrity of the symposia; however, verbatim reproductions of previously published papers are not accepted. Both reviews and reports of research are acceptable since symposia may embrace both types of presentation.

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

PREFACE early thirty centuries ago Homer referred to "pest-averting sulfur." Today many organosulfur natural products are known to have pesticidal activity, and more than one-third of the total number of synthetic organic pesticides contain sulfur. Pesticides are categorized commonly as organochlorine or organophosphorus compounds but rarely as organosulfur compounds. This is caused in part by the heterogeneous nature of the organosulfur pesticides in contributing to the biological activity.

N

r

Pesticide effectiveness and selectivity often are increased by using apolar and relatively stable compounds which can be converted metabolically to the active toxicants. Most sulfur-containing pesticides are in fact propesticides undergoing metabolic activation by reactions involving or initiated by oxidation. These sulfoxidations increase the reactivity of phosphorothionate insecticides as phosphorylating agents and of thiocarbamate herbicides as carbamoylating agents. Metabolic conversion of sulfides to sulfoxides and sulfones also alters the reactivity, solubility, and ease of translocation of systemic pesticides. Thus, introduction of a sulfur-containing moiety may enhance the selectivity, sometimes with a reduction in mammalian toxicity. Endogenous sulfur compounds play a critical role in the action of many insecticides, fungicides, and herbicides. The primary target enzyme or receptor may have an essential thiol group and secondary target sites also may have such groupings. The metabolism may be dominated by glutathione-dependent processes thereby allowing interactions with synergists or antidotes that alter the efficiency of these sulfur reactions and thus the toxicity of the pesticides. Recognition of the importance of sulfur chemistry and biochemistry in relation to pesticide metabolism and action led to a symposium with this title at the August 1980 National Meeting of the American Chemical Society. This symposium volume considers some principles of sulfur chemistry as they affect our understanding of sulfur-containing compounds in biological systems. Expanding knowledge of organosulfur chemistry ix

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

and biochemistry will influence significantly the design of pesticides for the future and the evaluation of conditions for the safe and efficient use of these pest control chemicals.

Joseph D. Rosen Department of Food Science Cook College Rutgers University New Brunswick, NJ 08903

PHILIP S. MAGEE

Agricultural Chemicals Division Chevron Chemical Company 940 Hensley St. Richmond, CA 94804

John E. Casida Pesticide Chemistry and Toxicology Laboratory Department of Entomological Sciences University of California Berkeley, CA 94720 February, 1981

x In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

1 Low-Molecular-Weight Organosulfur Compounds in Nature: The Search for New Pesticides ERIC BLOCK Department of Chemistry, University of Missouri, St. Louis, MO

63121

A rich variety of organosulfur compounds are found in living systems. A list of biochemicall would include, among others and methionine, peptides such as glutathione, and the recently characterized biologically extremely potent glutathione deriva­ tive leukotriene C-1 (1)(1)(Fig. 1) (the "slow reacting substance" of anaphylaxis (SRS) which is known for its role in the lungs during asthma attacks), polycyclic peptide antibiotics such as bacitracin, gliotoxin, cephalosporin, penicillin, and the recently discovered β-lactam antibiotic thienamycin (2)(2), cofactors and vitamins such as thiamine, biotin, coenzyme A and α-lipoic acid, a bio­ logical alkylating agent, S-adenosylmethionine, the biological redox systems ferredoxin and rubredoxin and sulfur-containing bases found in bacterial transfer-RNA such as 4-thiouracil (3). A remarkable range of simpler organosulfur compounds are known to be widely distributed throughout the plant kingdom (3,4,5,6). These compounds often make important contributions to the odor and flavor of many of the common comestibles. In some instances these sulfur compounds may also serve by their odor and taste to repel predators or to act for the plant as resistance factors against infection by microorganisms (e.g. as natural pest­ icides). Not to be outdone, some insects such as the onionmaggot, Hylemya antiqua, and t u r n i p (or vegetable) w e e v i l , Listroderes obliquas, have evolved so that some of the same organosulfur compounds a c t as a t t r a c t a n t s and s t i m u l a t e egg l a y i n g and b i t i n g

(Z) A number of i n s e c t s and higher animals have a l s o been found to possess unusual small organosulfur molecules which may serve as defensive s e c r e t i o n s , sex a t t r a c t a n t s , or scent markers · In many cases the low molecular weight organosulfur compounds occur i n p l a n t s (and p o s s i b l y i n animals as w e l l ) i n an odorless com­ bined form (e.g., as peptides o r g l y c o s i d e s ) and a r e r e l e a s e d enzymatically when the t i s s u e i s i n j u r e d or s t i m u l a t e d , i . e . during a t t a c k by a predator. F i n a l l y i t should be noted that c e r t a i n s u l f u r compounds reported to be of n a t u r a l o r i g i n may 0097-6156/81/015 8-0003$05.00/0 © 1981 American Chemical Society

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

SULFUR IN PESTICIDE ACTION AND METABOLISM

4

be a r t i f a c t s formed during the i s o l a t i o n procedures through r e a c t i o n s of unstable p r e c u r s o r s . In the survey of n a t u r a l organosulfur molecules that follows I have focused on those lower molecular weight compounds known to show b i o l o g i c a l a c t i v i t y (or which might be expected to possess such a c t i v i t y ) , p a r t i c u l a r l y as p e s t i c i d e s and a n t i b i o t i c s , and have emphasized the more current l i t e r a t u r e with coverage through J u l y 1980. There are v a l u a b l e lessons f o r us to l e a r n from a c a r e f u l study of attempts by other species to use chemicals as pest c o n t r o l agents i n t h e i r s t r u g g l e f o r s u r v i v a l . A few i l l u ­ s t r a t i o n s w i l l be given showing how t h i s knowledge has been put i n t o p r a c t i c e i n the design of new p e s t i c i d e s . I t i s convenient to group the compounds according to whether the s u l f u r i s bonded i n an a c y c l i c or h e t e r o c y c l i c manner. The a c y c l i c n a t u r a l l y o c c u r r i n g organosulfur compounds w i l l be discussed f i r s t . A c y c l i c S u l f u r Systems Plant Sources. The t u r n i p (Brassica campestris), rutabaga (Brassica napus), cabbage (Brassica oleracea) r a d i s h (Raphanus sativus) and a number of other p l a n t s contain 2-phenylethylisothiocyanate, PhCH CH NCS, (3), while the r a d i s h , b l a c k mustard (Brassica nigra) and penny~cress (Thlaspi arvense) contain related isothiocyanates which possess sharp i r r i t a t i n g odors and cause b l i s t e r i n g of the s k i n . C e r t a i n of these i s o t h i o c y a n t e s such as 3 are i n s e c t i c i d a l or a n t i b i o t i c p r o t e c t i n g the p l a n t against p a r a s i t e s (6^, 8_). At the same time a l l y l i s o t h i o c y a n a t e , CH2=CHCH2NCS, found i n v a r i o u s Brassica s p e c i e s , has been found to be an a t t r a c t a n t f o r the vegetable w e e v i l , Listroderes efoliquus which feeds on B r a s s i c a species and the f l e a b e t t l e , Phyllotreta cmciferae and to i n i t i a t e b i t i n g by the former harmful pest (7) · The i s o t h i o c y a n a t e s , or mustard o i l s as they are c a l l e d , have been known s i n c e the nineteenth century to be secondary products a r i s i n g from breakdown of mustard o i l glucosides (widely d i s t r i b u t e d i n the C r u c i f e r a e family) when c e l l u l a r s t r u c t u r e i s d i s r u p t e d (6)· The Allium species such as the onion (Allium cepa), g a r l i c (Allium sativum)^ s h a l l o t (Allium ascalonicum), chive (Allium scordoprasum), caucas (Allium victorialis)> and leek (Allium odonon) and other members of the L i l i a c e a e f a m i l y are r i c h n a t u r a l sources of b i o l o g i c a l l y a c t i v e organosulfur compounds. As i n the case of the mustard o i l from B r a s s i c a s p e c i e s , the organosulfur compounds from A l l i u m species are found i n the plant t i s s u e s i n an odorless combined form, e.g. as γ-glutamyl peptides of the S - a l k y l - and S-alkenylcysteine s u l f o x i d e s 4, F i g u r e 2. A l l i i n , (R = H, R = CH =CH) the n a t i v e c o n s t i t u e n t of g a r l i c i s broken down enzymatic a l l y to a l l i c i n , CH =CHCH S(0)SCH CH=CH (6c), when the p l a n t i s crushed. A l l i c i n i s b a c t e r i o c i d a l and along~with t h i o s u l f i n a t e s 5a and 5b from onion or g a r l i c possesses a n t i f u n g a l , a n t i v i r a l and tumor i n h i b i t i n g a c t i v i t y (9). D i a l l y l d i s u l f i d e , (CHf=CHCH S) , and d i a l l y l t r i s u l f i d e , (CH =CHCH S) S, two other components of 3

2

2

Λ

9

>

f

2

2

2

2

2

2

2

2

2

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

2

1.

BLOCK

5

Low-Molecular-Weight Organosulfur Compounds

H

OH Λ00Η

/

C5H11

If

*CH2CHC0NHCH2C00H NHC0CH2CH2CHC00H NH 2

r^-SCH2CH2NH

2

COOH

Figure 1.

RR'CHS(0)CH 2 CH(NH 2 )COOH

Leukotriene C-l (1) and thienamycin (2)

ALLIINASE PYRIDOXAL

RR'CH-S-O-H

+

CH 2 =C(NH 2 )C00H

PHOSPHATE

R' RR'CHS(0)SCHRR' 6 A

R = R'

Β

R = H,

= H R'

= C2H5

c

R = H,

R'

= CH2=CH

W / Y 7 A

R = C2H5,

R'

= H

(MAJOR) Β

R = H,

R'

= C2H5

(MINOR)

Figure 2. Organosulfur compounds from allium species

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

6

SULFUR IN PESTICIDE ACTION AND METABOLISM

g a r l i c o i l have been found to possess l a r v i c i d a l a c t i v i t y ( f a t a l at 5 ppm t o Culex pipiens, a mosquitoe s p e c i e s ) (10). The i n t e n s e l y lachrymatory p r i n c i p l e of the onion, shown to be a 20:1 mixture of Z- and E - p r o p a n e t h i a l S-oxide, 7a and 7b, (1J., 12, 13) i s d e r i v e d by enzymatic breakdown of trans-(+)-S-(1-propeny1)-Lc y s t e i n e s u l f o x i d e CH CH=CHS(0)CH CH(NH )COOH f o l l o w e d by r e arrangement of the i n t e r m e d i a t e ( E ) - l - p r o p e n e s u l f e n i c a c i d , CH3CH=CHS-0-H (ljL, 13). The onion lachrymator may serve an ecol o g i c a l f u n c t i o n i n r e p e l l i n g some h e r b i v o r e s or p a r a s i t e s . D i n-propyl d i s u l f i d e and other d i s u l f i d e s produced by the onion a c t as powerful a t t r a c t a n t s f o r the onion maggot (Hylemya antiqua), and b l a c k b l o w f l y (Phormia regina)(only the female i s a t t r a c t e d ! ) (_7) and s t i m u l a t e egg l a y i n g by the onion maggot and leek moth (Acrolepiopsis asseotella) (14). Crude onion j u i c e i n h i b i t s the growth of Escherichia coli, Pseudomonas pyocyaneus, Salmonella typhi and Bacillus subtilis R e c e n t l y , the c a u s a t i v d e r m a t i t i s known as "Dogger Bank i t c h " has b e e n i d e n t i f i e d as the (2-hydroxyethyl)dimethylsulfoxonium i o n , (CH )2S(0)CH2CH20H, produced by the marine bryozoan, Alcyonidium gelatinosvm (16). Related s a l t s are known such as 6-dimethylsulfonium pentanoic a c i d , (CH ) *CH2CH2CH2CH2C02H, from the c o a s t a l dune p l a n t Diplotaxis tenuifolia, and t h e t i n , (CH ) SCH2CH2C02H, a common component of v a r i o u s marine algae (17 ). A v a r i e t y of other compounds c o n t a i n i n g a c y c l i c s u l f u r have r e c e n t l y been i s o l a t e d from p l a n t s although nothing has been reported on t h e i r b i o l o g i c a l a c t i v i t y . These examples would i n c l u d e d i e t h y l t e t r a s u l f i d e , (C2H SS)2, and r e l a t e d p o l y s u l f i d e s and h y d r o s u l f i d e s from the f r u i t of the d u r i a n , Durio zibethinus (a much c h e r i s h e d south-east A s i a n f r u i t ) ( 1 8 ) , germacradienolide t h i o l 8 from Eupatorium mikanioides ( 1 9 ) , sesquiterpene l a c t o n e su I f one g from the r o o t s of Eelenium autvamale ( 2 0 ) , v i n y l s u l f i d e s 10 and 11 ( S - p e t a s i n ) from Petasites japonicus and Petasites nybridus^ r e s p e c t i v e l y (21, 22), t h i o s u l f o n a t e 12, from the mushroom Lentinus edodes, and S-methylthiomethyl 2-methylbutanet h i o a t e , C2H CH(CH )C(0)SCH SCH , from the e s s e n t i a l o i l of hops (23). (See F i g u r e 3.) 3

2

2

+

3

3

2

3

2

5

5

3

2

3

Animal Sources. The spray of the s t r i p e d skunk (Mephitis mephitis) which i n c l u d e s t r a n s - 2 - b u t e n e t h i o l , CH CH=CHCH SH, 3m e t h y l b u t a n e t h i o l , (CH ) CHCH2CH SH, and c r o t y l methyl d i s u l f i d e , CH CH=CHCH SSCH (24) i s o b v i o u s l y v e r y e f f e c t i v e i n r e p e l l i n g (and making q u i t e m i s e r a b l e ) would-be p r e d a t o r s . As an a s i d e i t may be noted t h a t the s e n s i t i v i t y of the human nose to simple t h i o l s ( c a . 0.02 ppb of methanethiol and 0.0007 ppb of 2-methyl2 - b u t a n e t h i o l can be detected (25)) and other s m a l l s u l f u r molecules as w e l l as to low molecular weight amines coupled w i t h the f a c t t h a t many animals a l s o f i n d t h e i r s m e l l o f f e n s i v e supports s p e c u l a t i o n t h a t n a t u r a l s e l e c t i o n developed t h i s o l f a c t o r y sens i t i v i t y as a form of p r o t e c t i o n f o r the organism a g a i n s t the 3

3

3

2

2

2

2

3

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

1.

BLOCK

LoW'Molecular-Weight Organosulfur Compounds

CH 3 S(0)2SCH20CH 2 SCH 2 S(0)CH3

12

Figure 3. Compounds containing acyclic sulfur

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

7

SULFUR IN PESTICIDE ACTION AND METABOLISM

8

i n g e s t i o n of decaying food (low molecular weight organosulfur compounds and amines are a l s o products of b i o l o g i c a l decay). A p r a c t i c a l a p p l i c a t i o n of t h i s o b s e r v a t i o n i s the use of d i a l l y l d i s u l f i d e , (CH =CHCH S) , as a r e p e l l e n t f o r i n j u r i o u s b i r d s (26)» The occurrence of Δ -isopentenyl methyl s u l f i d e , CH =C(CH )CH CH SCH , and 2-phenylethyl m e t h y l s u l f i d e , PhCH CH SCH3, as components of the u r i n e scent mark of the red fox (Vulpes vulpes) (27), of 5-methylthio-2,3-pentanedione, CH C(0)C(0)CH CH SCH , as a v o l a t i l e s e c r e t i o n from the a n a l scent gland of the s t r i p e d hyena (Hyaena hyaena) (28), of d i ( 3 - m e t h y l b u t y l ) s u l f i d e , ((CH ) CHCH CH ) S, from the polecat (Mustela putovius) (29), and of dimethyl d i s u l f i d e , CH SSCH , as an a t t r a c t a n t pheromone i n the v a g i n a l s e c r e t i o n of the hamster (Cricetus cricetus) (30) provide examples of organosulfur compounds i n v o l v e d i n chemical i n t e r a c t i o n s between animals. 2

2

2

3

2

3

2

3

2

2

3

2

2

2

3

3

2

2

2

2

3

3

H e t e r o c y c l i c S u l f u r System P l a n t Sources, F i g . 4 d i s p l a y s a broad range of organo­ s u l f u r compounds found i n p l a n t s or animals i n which the s u l f u r atom(s) i s p a r t of a r i n g . Many of these compounds show sub­ s t a n t i a l b i o l o g i c a l a c t i v i t y . Thus, v a r i o u s s p e c i e s of marigold (Tagetes) and other members of the Compositae f a m i l y c o n t a i n a - t e r t h i e n y l , 21, and 5 - ( 3 - b u t e n - l - y n y l ) - 2 , 2 ' - b i t h i e n y l , 20, which are l e t h a l to b a c t e r i a , yeasts and other f u n g i , nematodes and f i s h i n near-UV l i g h t (31,32,33). In the o r i g i n a l study i n t h i s a r e a , 24 kg of marigold r o o t s were processed to u l t i m a t e l y a f f o r d 200 mg of 21 which proved to be n e m a t i c i d a l against Heterodera rostochiensis l a r v a e , Pratylenohus penetrans, Ditylenchus dipsaci and other species of nematodes (34). Other n a t u r a l t h i o phene r i n g c o n t a i n i n g nematicides i n c l u d e 2-phenyl-5-(l -propynyl)thiophene 19 from Coreopsis lanceolata (35) and r e l a t e d compounds from ChrystTumthenum vulgare (36) and other species (37). A second group of b i o l o g i c a l l y a c t i v e molecules i s o l a t e d from n a t u r a l sources are 1 , 2 - d i t h i o l a n e d e r i v a t i v e s 22-25 which are s t r u c t u r a l l y r e l a t e d to the c o f a c t o r , α-lipoic a c i d . Asparagusic a c i d , 22, present to the extent of about 35 ppm i n the r o o t s of asparagus (Asparagus officinalis) i s a c t i v e against the p l a n t p a r a s i t i c nematodes Paratylenchus penetrana and P. eurvitatus, Eeterodera rostochiensis and #. glycines, and Meloidogyne hapla, and i s considered t o be a major f a c t o r i n n a t u r a l r e s i s t ­ ance of asparagus (38). Asparagusic a c i d , i t s syn and a n t i Soxide (39) and some r e l a t e d a c y c l i c d e r i v a t i v e s are a l s o very e f f e c t i v e p l a n t growth i n h i b i t o r s . Compounds 23 (4-methylthio1 , 2 - d i t h i o l a n e ) and 32 ( 5 - m e t h y l t h i o - l , 2 , 3 - t r i t h i o l a n e ) are r e s p o n s i b l e f o r the rank, pungent s m e l l a s s o c i a t e d w i t h the stonewort (Chara globularis). Whenever t h i s green a l g a occurs i n a pond i t dominates the a l g a l f l o r a of the ecosystem, apparently because 23 and 32 act as extremely a c t i v e i n h i b i t o r s of photosyn­ t h e s i s , thus eliminating'would-be competitive species (40). The f

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

1.

BLOCK

Low-Molecular-Weight Organosulfur Compounds

Figure 4.

Naturally occurring thiaheterocycles

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

9

SULFUR IN PESTICIDE ACTION AND METABOLISM

10

p r o p e r t i e s of 1,2-dithiolanes 24 and 25 w i l l be discussed below. A number of unusual c y c l i c p o l y s u l f i d e s showing a n t i b i o t i c a c t i v i t y against b a c t e r i a and fungi have been i s o l a t e d from the red alga Chondria oalifomica (41) and from the mushroom Lentinus edodes (42). Both 34 (1,2,4,6-tetrathiepane) and 35 (1,2,3,5,6pentathiepane or l e n t h i o n i n e ) have been i s o l a t e d from both sources, while 1,2,3,4,5,6 -hexathiepane, 36, was obtained from the mushroom and 27 ( 1 , 2 , 4 - t r i t h i o l a n e ) , 2g ( 1 , 2 , 4 - t r i t h i o l a n e 4-oxide), 30 ( 1 , 2 , 4 - t r i t h i o l a n e 1-oxide), 37 (1,2,4,6-tetrathiepane 4,4dioxide) and 40 (1,2,4,5,7,8,10,11-octathiacyclododecane) were obtained from the alga ( f o r a synthesis of 29, see (43); f o r s t u dies on precursors to 35, see (44)). T r i t h i o l a n e s r e l a t e d to 27, 2g, and 30 have been i s o l a t e d from steam v o l a t i l e o i l of onion (e.g. 28) (45) and root m a t e r i a l of Petivevia alliaoeae (46). Other b i o l o g i c a l l y a c t i v e n a t u r a l l y derived t h i a h e t e r o c y c l e s include ôjô'-dihydroxythiobinupharidine spatterdock or yellow pon v i r i d i n , 39, an a n t i o b i o t i c from Streptomyces griseus (49) and the a n t i b i o t i c u r e o t h r i c i n 26 (50). Other n a t u r a l t h i a h e t e r o c y c l e s whose b i o l o g i c a l a c t i v i t i e s have not yet been reported include l-cyano-2,3-epithiopropane, 13, from cabbage, (51) the isomeric 2-methyl-4-propyl-l,3-oxathianes, 31, from the Hawaiian yellow passion f r u i t (Passiflora edulis) (52), the sesquiterpene m i n t s u l f i d e , 18, from o i l of peppermint (Mentha piperita) (53), the d i t e r p e n o i d , p h a r b i t i c a c i d , 16, from the seeds of the Japanese morning-glory (Pharbitis niV) (thought to be a g i b b e r e l l i n A r e g u l a t o r ) (54), t r a n s - 3 , 4 - d i e t h y l - l , 2 - d i t h i e t a n e 1,1-dioxide, 15, formed by d i m e r i z a t i o n of the lachrymatory f a c t o r of the onion, Z- and E-propanethial S-oxide 7a,b (5J5), and d i s u l f i d e 33 from two brown algae of the genus Diotyopteris (56,57). Animal Sources. The marine a n n e l i d worm Lumbrineris heteropoda produces an i n s e c t t o x i n n e r e i s t o x i n , 24, 4-N,N-dimethy1amino-l,2-dithiolane (5-0( 2 - c h l o r o a l l y l ) thiocarbamate s u l f e n a t e e s t e r s (10) i n q u a n t i t a ­ t i v e y i e l d (7). This thermal rearrangement i s analogous to the r e v e r s i b l e rearrangement of £-tolyl a l l y l s u l f o x i d e s (12, 13). A s u l f e n a t e e s t e r analogous to 10 but with methyl instead of c h l o r i n e i s formed on MCPBA o x i d a t i o n of the corresponding Sm e t h a l l y l thiocarbamate; t h i s patent report (14) does not mention any intermediates or speculate on the mechanism of the r e a c t i o n . Formation of the s u l f e n a t e e s t e r i s conveniently monitored not only by IR and NMR but a l s o by CI-MS, i n the l a t t e r case because i n contrast to the s u l f o x i d e s (7, 9) the s u l f e n a t e s are s u f f i c i e n t l y s t a b l e to e x h i b i t a strong molecular i o n (7).

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

5.

SCHUPHAN ET AL.

S-Chloroallyl Thio- and Dithiocarbamate Herbicides 69

S ^ ( 3 - C h l o r o a l l y l ) thiocarbamate s u l f o x i d e s Ce.£., 5-7) undoubtedly rearrange i n an analogous manner but i n t h i s case the s u l f e n a t e q u i c k l y undergoes an a d d i t i o n a l 1,2-éliminâtion r e a c t i o n (7) . The r e s u l t i n g products are the Ν,Ν-dialkylcarbamoyls u l f e n y l c h l o r i d e (11) and the carbonyl compound, i . . ^ . aldehydes from the 3 - c h l o r o a l I y l d e r i v a t i v e s (e.£., 5 and 6) and a c i d c h l o ­ r i d e s i n the case of the 3,3-dichloro analogs (e.&., 7) (7,8). This thermal rearrangement along with the 1,2-elimination r e a c ­ t i o n i s analogous to a sequence p r e v i o u s l y reported f o r a r y l 3-chloroallyl sulfoxide

11

2-chloroacrolein

Oxidations. The Stalky1 and S^-benzyl thiocarbamate s u l f o x ­ ides are converted almost q u a n t i t a t i v e l y to the corresponding sulfones on MCPBA o x i d a t i o n i n chloroform (3-5). S^(2-Chloroa l l y l ) thiocarbamate 12 or the corresponding s u l f o x i d e (4) w i t h excess oxidant gives the s u l f o n e d e r i v a t i v e (13) which i s i s o ­ meric with the s u l f i n a t e e s t e r (14) obtained from the s u l f e n a t e e s t e r (10) with 1 mole of MCPBA. Compounds 13 and 14 give c h a r a c t e r i s t i c NMR (Table II) and CI-MS spectra (see below). The unusual NMR s p e c t r a l feature of the S ^ ( 2 - c h l o r o a l l y l ) t h i o c a r b a ­ mate s u l f o x i d e (4) i n e x h i b i t i n g a s i n g l e t f o r the two protons of the terminal methylene i s r e t a i n e d on f u r t h e r o x i d a t i o n to the sulfone (13) (Tables I and I I ) . Oxidation of s u l f e n a t e e s t e r 10 r e s u l t s i n the aforementioned s p l i t t i n g of the CH3 and CH2-N proton s i g n a l s due to the c h i r a l center and r e s t r i c t e d r o t a t i o n around the amide C-N bond of the s u l f i n a t e e s t e r (14) (Table I I ) . Both 13 and 14 give a molecular i o n on CI-MS, but sulfone 13 decomposes by l o s s of ethylene and s u l f i n a t e 14 cleaves o f f oxy­ gen to give the base peak. Further o x i d a t i o n of 14 r e s u l t s i n a new product with completely d i s t i n c t NMR s i g n a l s c o n s i s t e n t with those expected f o r the s u l f o n a t e e s t e r (Table I I ) .

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

SULFUR IN PESTICIDE ACTION AND METABOLISM

70

TABLE I I 1. H Chemical S h i f t Data (ppm) f o r S - ( 2 - C h l o r o a l l y l ) N,N-Diethylthiocarbamate and I t s Oxidation Products. S o l u t i o n s i n CDCl^ a t 20°-40°C with t e t r a m e t h y l s i l a n e as the i n t e r n a l standard. Proton c o u p l i n g i n Hz: CH~-CH , 7.1; =CH , 1.1; OCH^ i n compound 14, 13.1. u b l e t , tt==t tr riipplleett,, qa =quartet, m=multiplet. s = s i n g l e t , d = doublet, = quart 2

Compound number and s t r u c t u r e

CH (tj

CH -N (qa)

S-CH (s)

1.18

3.40

3.84

3

^"Y

Nv^S^^X^j

1

2

2

2

0CH (s)

=CH, (d)'

2

5.26 5.48

1.26(m

0

0

13

J '

1

1.30(m) CI

10

\

N

s

^°v>

1.18

3.46 3.75

5.68(s)

4.39

3.18

4.48

5.45

*CI

14

3s^ | 0v

"^N^

1.23 1.29

3.47 3.58

4.46(d) 5.46 4.73(d) 5.57

1.25 1.30

3.44 3.70

4.92

0 0,

Ν

γ\Χ, S. Jl

U

γο- ^ c .

excess^

~Λ

!

7 ^

_yοY|^ , c

^

ς

N

-*

M+ l

240

[ M

-C H ] 2

4

+

,

2

I

13

.

N

II

N

12 \ / Nf^

5.51 5.66

y

10

/ ° \ 1 MCPBA JL

^ c ,

o \ \ , ^ ° \ ) S^ I N>

-J

M + l

S

A

τ

c

l

M

_

0

240 2

14

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

2

3

2

5.

SCHUPHAN ET AL.

Dithiocarbamate

S-Chloroallyl Thio- and Dithiocarbamate Herbicides 71

Sulfines

Synthesis. Oxidation of S - c h l o r o a l l y l dithiocarbamates with equimolar MCPBA i n chloroform, methylene c h l o r i d e or methanol a t -25°C gives a very exothermic r e a c t i o n l e a d i n g to the correspond­ ing dithiocarbamate s u l f i n e s (15), i^.e. s u l f a l l a t e s u l f i n e (16) and i t s t r a n s - 2 , 3 - d i c h l o r o analog (17) (16). Cl>.

>Y N S

s

>Y ^c, s

s

^o

15

^o 16

Ζ/ γ^ο, Ν

s

^o 17

Thus, the p r e f e r r e d s i t e of p e r a c i d o x i d a t i o n i s the thiono s u l f u r i n dithiocarbamates (16), as with d i t h i o e s t e r s (17, 18), r a t h e r than the t h i o l o s u l f u r S p e c t r a l Features. Examination of the equimolar s u l f a l l a t e MCPBA r e a c t i o n mixture r e v e a l s a product with a CI-MS peak appropriate f o r s u l f a l l a t e p l u s one oxygen (16). NMR s p e c t r a l data (Table I I I ) e s t a b l i s h that the p e r a c i d monooxygenation products of s u l f a l l a t e and c h l o r o - s u l f a l l a t e a r e s u l f i n e s 16 and Γ7 (16). There i s an u p f i e l d s h i f t of ~0.9 ppm f o r the S-CH protons a t t r i b u t a b l e to "through space" s h i e l d i n g by the negative environment of the s u l f i n e oxygen. Oxidation a t the t h i o l o instead of the thiono s u l f u r would have l e d to a downfield s h i f t f o r the S-CH protons, 3.84 ppm f o r the corresponding thiocarbamate (12) and 3.90 ppm f o r i t s s u l f o x i d e d e r i v a t i v e (4) (Table I I ) . S i m i l a r s p e c t r a l changes occur on conversion of d i t h i o e s t e r s to t h e i r s u l f i n e d e r i v a t i v e s (17). I t appears l i k e l y that s u l f i n e s 1J3 and Γ7 are obtained as i s o m e r i c a l l y - p u r e m a t e r i a l s s i n c e only a s i n g l e S-CH s i g n a l i s evident (Table I I I ) . The l a c k of s i g n i f i c a n t changes i n the chemical s h i f t s of the N-Ct^CH^ protons i n c o n t r a s t to the S-CH^ protons on o x i d a t i o n of s u l f a l l ­ ate and c h l o r o - s u l f a l l a t e (Table I I I ) provides supporting evidence f o r the Z^-sulfines. 2

2

2

Rearrangements. P e r a c i d monooxygenation of dithiocarbamates with 3 - c h l o r o a l l y l s u b s t i t u e n t s (e , chloro-sulfallate), i n c o n t r a s t to the analogous thiocarbamates (e.&., d i a l l a t e ) , does not lead to [2,3] sigmatropic rearrangement followed by 1,2e l i m i n a t i o n r e a c t i o n s , i.e. the equimolar chloro-sulfallate-MCPBA r e a c t i o n gives no products with NMR s i g n a l s f o r =CH protons (16). T h i s lends support to other NMR s p e c t r a l evidence noted above that only the thiono group i s o x i d i z e d . 2

S u l f i n e s 16^ and JL7 a r e r e l a t i v e l y s h o r t - l i v e d i n s o l u t i o n , r e v e r t i n g to the s t a r t i n g m a t e r i a l s w i t h i n a few hours a t 40°C or r a p i d l y on a d d i t i o n of triphenylphosphine or attempted i s o l a t i o n

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

4.31

5.33 5.60

3 (s)

4/5 (d)

a

Singlet.

Complex obtained on r e a c t i o n of

New (s)

3.87 3.96

3.77 4.03

2 (qa)

10.06

5.46 5.94

4.48

3.76 4.15

1.44 1.48

a

Complex 22

6.31

4.52

3.38 4.00

1.30

b

Chlorosulfallate

1 2

l

6.43

3.69

3.88 3.95

1.29

b

Sulfine 17

S

C

^

Cl

10.03

6.41

4.74

3.79 4.16

b

t f a

1.46

M

Complex 22

s u l f a l l a t e or c h l o r o -• s u l f a l l a t e with 4 molar e q u i v a l e n t s of MCPBA.

5.30 5.54

3.43

1.29

Sulfine 16

H ^ H

1.28

Sulfallate

2

1 (t)

Protons

1

3

*H Chemical S h i f t Data (ppm) f o r S u l f a l l a t e , C h l o r o - S u l f a l l a t e , T h e i r S u l f i n e D e r i v a t i v e s and Further Oxidation Products. S o l u t i o n s i n CDC1 at 20°-40°C with t e t r a m e t h y l s i l a n e as the i n t e r n a l standard. Proton coupling i n Hz: CH^-CH^, 7.1; = CH^, 1.1.s = s i n g l e t , d = doublet, t = t r i p l e t , qa = quartet.

TABLE I I I

Ο

>

w H

> α

W > Ο H δ

Β

H Ο

C/3

w

to

5. SCHUPHAN ET AL.

S-Chlorocillyl Thio- and Dithiocarbamate Herbicides 73

i n v o l v i n g e x t r a c t i o n with aqueous sodium carbonate s o l u t i o n (16). This l o s s of oxygen probably i n v o l v e s an o x a t h i i r a n intermediate 18 which can a l s o extrude s u l f u r . D e s u l f u r a t i o n to form the t h i o ­ carbamate (e.j>. 1 2 ) i s normally a minor pathway but becomes major when p - t o l u e n e s u l f o n i c a c i d i s present i n the r e a c t i o n mixture (16). R

MCPBA

\

15

Y"

-LsJ

18

Oxidations. Treatment of s u l f a l l a t e or c h l o r o - s u l f a l l a t e with a 3-5-fold molar excess of MCPBA leads to e n t i r e l y d i f f e r e n t products than equimolar MCPBA. This i s evident on comparing the NMR s p e c t r a l features of s u l f a l l a t e , s u l f i n e 16, and complex 22 or of the

MCPBA

2 MCPBA CI S

sulfallate

19

u Ov^A

^0

Τ * u

Ύ 0

V

21

20

22

Γ N N ^ H

Τ

Τ S

+ CI^^ ~

S

v

0

1

Q -^CI

0

+ Ar^O^Ar

0

23

24

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

SULFUR IN PESTICIDE ACTION AND METABOLISM

74

analogous products from c h l o r o - s u l f a l l a t e (Table I I I ) . Complex 22^ gives diethylformamide (23), 2 - c h l o r o a l l y l d i s u l f i d e (24) and the anhydride of m-chlorobenzoic a c i d on TLC i s o l a t i o n u s i n g s i l i c a g e l or on treatment with p y r i d i n e , h y d r o c h l o r i c a c i d , sod­ ium b i s u l f i t e or sodium carbonate. However, complex 22^ gives com­ pletely different H (Table I I I ) and C NMR s i g n a l s than any one or combination o f a l l of the compounds obtained on TLC i s o l a t i o n (16) . The s t r u c t u r e i n d i c a t e d f o r 22 i s one way to r a t i o n a l i z e i t s s p e c t r a l f e a t u r e s and r e a c t i o n c h a r a c t e r i s t i c s (16). NMR s t u d i e s r e v e a l that the low r e s o n a t i n g proton ( - 10 ppm) i s d i r e c t l y bonded to a carbon (181.2 ppm) which i s s p l i t to two l i n e s i n the l ^ C proton o f f resonance decoupling mode. T h i s o b s e r v a t i o n i s c o n s i s t e n t with a s u l f i n e carbon bearing one proton, which i n d i ­ cates that the dithiocarbamate molecule i s already cleaved i n solution. S u l f i n e s u l f o x i d e JL9 i s a p p r o p r i a t e f o r the cleavage r e a c t i o n due to i t s e x c e l l e n sulfide i n d i c a t e s tha complex i s a s u l f o x i d e r a t h e r than a s u l f o n e . A p o r t i o n of the low temperature NMR spectrum of complex 22 i s very s i m i l a r i n a l l r e s p e c t s y e t not i d e n t i c a l with that of diethylthioformamide s u l f i n e (20) (prepared by MCPBA o x i d a t i o n of the thioformamide a t -30°C). Thioformamide s u l f i n e 2£ decomposes very f a s t above -20°C to give diethylformamide (23) and elemental s u l f u r . Accordingly, s u l f i n e 20 must e x i s t i n s o l u t i o n i n a complexed form. A l l p r o p e r t i e s of complex 22 from s u l f a l l a t e and the analogous complex "22" from c h l o r o - s u l f a l l a t e a r e c o n s i s t e n t with the i n d i c a t e d adduct of diethylthioformamide s u l f i n e (20) and a mixed peroxyanhydride (21). 1 3

Other o x i d a t i o n products of s u l f a l l a t e a r e formed v i a t h i o carbamate 12^ as d i s c u s s e d b e f o r e . Metabolism of T h i o - and

Dithiocarbamates

Oxidations and Rearrangements. S - A l k y l and S-benzyl N,Nd i a l k y l t h i o c a r b a m a t e s a r e converted to t h e i r s u l f o x i d e d e r i v a t i v e s (3) both i n v i v o i n r a t s and on i n c u b a t i o n with l i v e r microsomes and NADPH (3-5, 19-21). Studies with EPTC (25) r e v e a l that they may a l s o undergo h y d r o x y l a t i o n a t each a l k y l carbon (designated by arrows) and that carbon h y d r o x y l a t i o n a t the S-CI^ moiety gives an unstable intermediate which y i e l d s acetaldehyde on decomposi­ t i o n (19).

EPTC (25)

S - α —hydroxy compound

Although d i a l l a t e s u l f o x i d e (6) i s too unstable f o r p o s s i b l e i s o ­ l a t i o n as a metabolite, i t i s e s t a b l i s h e d as an i n v i t r o and

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

5.

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S-Chloroalîyî Thio- and Dithiocarbamate Herbicides 75

i n v i v o intermediate i n mammals by the d e t e c t i o n of two of i t s d e r i v a t i v e s , 1.e., the g l u t a t h i o n e (GSH) conjugate and i t s f u r t h e r metabolites formed by an i n i t i a l carbamylation r e a c t i o n (10) (see below) and 2 - c h l o r o a c r o l e i n detected i n the microsome-NADPH system and d e r i v e d from the rearrangement-elimination r e a c t i o n sequence discussed above (6). S u l f a l l a t e a l s o y i e l d s 2 - c h l o r o a c r o l e i n i n the microsome-NADPH system, presumably by JS-C^ h y d r o x y l a t i o n (22) on analogy with the metabolism of EPTC shown p r e v i o u s l y . Carbamylation Reactions. S - A l k y l , ^ - b e n z y l and S - c h l o r o a l l y l thiocarbamates do not r e a d i l y r e a c t with GSH. In c o n t r a s t , t h e i r s u l f o x i d e d e r i v a t i v e s Q and 6) are very e f f e c t i v e carbamylating agents f o r many t h i o l s i n c l u d i n g GSH (19, 21). The GSH conjugates formed i n v i v o v i a 3^ and j> are q u i c k l y cleaved, a c e t y l a t e d and f u r t h e r metabolized as f o l l o w s (19-21 23 24)

The s u l f e n i c a c i d l i b e r a t e d on carbamylation i s o x i d i z e d i n p a r t to the corresponding s u l f o n i c a c i d based on s t u d i e s with d i a l l a t e (6, 8 ) . T o x i c o l o g i c a l S i g n i f i c a n c e of O x i d a t i o n and Rearrangement Reactions P r o h e r b i c i d e s . Thio- and dithiocarbamates probably r e q u i r e metabolic a c t i v a t i o n p r i o r to e x e r t i n g t h e i r h e r b i c i d a l e f f e c t s . S u l f o x i d e metabolites of t h e S - a l k y l thiocarbamates are g e n e r a l l y more potent h e r b i c i d e s than the parent compounds (.3-5) . The h e r b i c i d a l a c t i o n of these s u l f o x i d e s probably r e s u l t s from t h e i r carbamylating a c t i o n f o r t h i o l s , although the s p e c i f i c t a r g e t s i t e or receptor i s not defined (23, 24). I t i s conceivable that the S - c h l o r o a l l y l thiocarbamate h e r b i c i d e s may a c t i n the same way, s i n c e t h e i r s u l f o x i d e s are a l s o potent carbamylating agents

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

SULFUR IN PESTICIDE ACTION AND METABOLISM

76

(8, 10). As an a l t e r n a t i v e , d i a l l a t e might be a p r o h e r b i c i d e and 2 - c h l o r o a c r o l e i n the u l t i m a t e h e r b i c i d e r e l e a s e d on decomposi­ t i o n of d i a l l a t e s u l f o x i d e (8). Promutagens. S - ( 2 - C h l o r o a l l y l ) t h i o - and dithiocarbamate h e r b i c i d e s , i n c o n t r a s t to the ^ - a l k y l thiocarbamates, show muta­ genic a c t i v i t y i n the Salmonella typhimurium "Ames assay; however, they are only mutagenic on metabolic a c t i v a t i o n , jL.e^, with the S9 mix (25-27). The requirement f o r the l i v e r microso­ mal mixed-function oxidase system f o r mutagenic a c t i v i t y l e d to the hypothesis that the S - c h l o r o a l l y l t h i o - and dithiocarbamates are promutagens and that an o x i d a t i o n process i s involved i n formation of the u l t i m a t e mutagens (6). I t was t h e r e f o r e of great i n t e r e s t to note that 2 - c h l o r o a c r o l e i n , an o x i d a t i v e metabolite of both d i a l l a t e and s u l f a l l a t e and i t s 2,3-dichloro and 2,3,3t r i c h l o r o analogs a r e Various c h l o r o a c r o l e i n mutagens formed from d i a l l a t e , t r i a l l a t e and s u l f a l l a t e as discussed l a t e r . Polymer formation occurs on r e a c t i o n of deoxyadenosine with the d i f u n c t i o n a l 2 - c h l o r o a c r o l e i n , probably due to cross l i n k i n g v i a S c h i f f base formation a t the carbonyl group and Michael a d d i t i o n at the double bond (28). 11

Balance of A c t i v a t i o n / D e t o x i f i c a t i o n Reactions. The a c t i v a t ­ ed intermediates or r e a c t i v e fragments appear to be carbamoyl s u l f o x i d e s or mono-, d i - and t r i c h l o r o a c r o l e i n s , a l l of which a r e r e l a t i v e l y unstable compounds. The carbamoyl s u l f o x i d e s a r e r a p i d l y d e t o x i f i e d by r e a c t i o n with GSH, i n v o l v i n g c a t a l y s i s by a GSH S-transferase i n the case of S - a l k y l and S-benzyl t h i o c a r b a ­ mate s u l f o x i d e s (3-5, 21, 23, 24) but probably not with S-chloro­ a l l y l thiocarbamate s u l f o x i d e s (6, 8^, 10). 2 - C h l o r o a c r o l e i n i s unstable i n metabolic systems i n c l u d i n g i n the presence of GSH (28). Highly r e a c t i v e a c t i v a t e d intermediates must a c t i n the same c e l l or even c e l l u l a r o r g a n e l l e i n which they a r e formed. Thus, compartmentalization phenomena may be important i n the a c t i o n of the m e t a b o l i c a l l y - a c t i v a t e d t h i o - and dithiocarbamates. Some Reactions of D i a l l a t e , T r i a l l a t e and S u l f a l l a t e of P o s s i b l e Importance to T h e i r Mutagenic and/or Carcinogenic P r o p e r t i e s . Current knowledge of the o x i d a t i o n and rearrangement r e a c t i o n s of S - ( 2 - c h l o r o a l l y l ) t h i o - and dithiocarbamate h e r b i ­ cides i n r e l a t i o n to t h e i r mutagenic a c t i v i t i e s i s i l l u s t r a t e d i n Figures 1 and 2. The mutagenesis data (6, 22, 28) i s from the Ames assay procedure (29) (Figure 3 ) . D i a l l a t e has been examined i n g r e a t e s t d e t a i l (Figure 1), i n part because i t gives the most potent mutagen(s) a f t e r a c t i v a t i o n [_ί·β.· a c t i v a t e d c i s - d i a l l a t e gives 40 revertants/nmole and a c t i v a t e d t r a n s - d i a l l a t e gives 25 revertants/nmole (Figure 3); t h i s potency i s s i m i l a r to that of the carcinogen benzo[a]pyrene 9

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

5.

SCHUPHAN ET AL.

S - a - h y d r o x y compound proximate mutagen

S-Chloroallyl Thio- and Dithiocarbamate Herbicides 11

2,3-dichloroacrolein

(0/-)

ultimate mutagen (104/-)

S — a — h y d r o x y compound p r o x i m a t e mutagen

2,3,3—trichloroacrolein

(0/-)

ultimate mutagen (224/-)

Figure 1. Oxidation and other reactions of diallate and triallate indicating genic activities of the products in the S. typhimurium TA 100 assay (revenants/ nanomole; without activation/with activation; / designates no data availab 2-Chloroacrolein is a diallate metabolite in the mouse liver microsome-NADP system. Dichloroallylsulfonic acid is a urinary metabolite of diallate. The o compounds are potential metabolites of the respective thiocarbamates. The carbamate sulfoxides are unstable at 25°C.

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

S

OH

mutagen

compound

Η

(113/74)

2-chloroacrolein ultimate mutagen y

OH (0/-)

10

CI

(0.03/0.11)

\

(0/0.45)

0

S/

YY °^

N

0·

13

0

/

N

S/0

I

14

^Y ^

0

_CI

(Ο/-)

H O - i ^ C I Ο

Figure 2. Oxidation and other reactions of sulfallate indicating mutagenic activities of the products in the S. typhimurium TA 100 assay (revertants/nmole; without activation/with activation; / desig­ nates no data available). All thio- and dithiocarbamates are formed from oxidations with MCPBA except for the α-hydroxy compound. 2-Chloroacrolein is a metabolite in the mouse liver microsomeNADPH system. The other compounds are potential metabolites. Several of the oxidized thio- and dithiocarbamates are unstable at 25°C.

proximate

S—α—hydroxy

12 (0/1.5)

Ο

4 (0/0.39)

ο

5.

SCHUPHAN ET AL.

S-Chloroallyl Thio- and Dithiocarbamate Herbicides 79

2500

20

40

60

80

nMoles

Figure 3. Mutagenic activities of the promutagens cis- and trans-diallate sulfallate, the proximate mutagen cis-diallate sulfoxide, and the ultimate mu 2-chloroacrolein, assayed with S. typhimurium strain TA 100 sensitive to base-pai substitution mutagens. The diallate isomers and sulfallate are not mutagenic w the S9 mix. S9 mix refers to a microsomal oxidase system prepared from ra and appropriate cofactors. The methodology is detailed in Refs. 6, 22, and

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

80

SULFUR IN PESTICIDE ACTION AND METABOLISM

(29)]. D i a l l a t e s u l f o x i d e (6) i s a very potent mutagen without metabolic a c t i v a t i o n and i t decomposes q u i c k l y i n t o 2-chloroa c r o l e i n , found to have the same mutagenic a c t i v i t y of 113 revertants/nmole i n assays without the S9 mix. D i a l l a t e metabo­ l i s m i n v o l v e s both d i a l l a t e s u l f o x i d e and 2 - c h l o r o a c r o l e i n as intermediates, as discussed above. Thus, d i a l l a t e gives a p r o x i ­ mate mutagen, the carbamoyl s u l f o x i d e , and an u l t i m a t e mutagen, 2 - c h l o r o a c r o l e i n , with about t h r e e - f o l d higher mutagenic a c t i v i t y than the b i o a c t i v a t e d c i s - d i a l l a t e . D i a l l a t e might a l s o y i e l d a second u l t i m a t e mutagen, the very potent 2 , 3 - d i c h l o r o a c r o l e i n , l i b e r a t e d f o l l o w i n g carbon h y d r o x y l a t i o n at the SJ-CI^ s i t e . T r i a l l a t e and s u l f a l l a t e are probably a c t i v a t e d to mutagenic metabolites from the c h l o r o a l l y l moieties without the involvement of t h e i r s u l f o x i d e s as the proximate mutagens (Figures 1-3). T r i a l l a t e s u l f o x i d e (T) Somewhat l e s s a c t i v e i t r i a l l a t e s u l f o x i d e on i t s rearrangement and e l i m i n a t i o n reac­ t i o n s . Hydroxylation of t r i a l l a t e at the carbon α to the s u l f u r p o t e n t i a l l y gives the h i g h l y potent mutagen 2 , 3 , 3 - t r i c h l o r o a c r o l e i n on decomposition of the ^-α-hydroxy proximate mutagen. S u l ­ f a l l a t e o x i d a t i o n with MCPBA does not y i e l d s u l f a l l a t e s u l f o x i d e or 2 - c h l o r o a c r o l e i n . Instead, i t gives a v a r i e t y of s u l f i n e s , s u l f o x i d e s , sulfones, S-O-sulfenate e s t e r s and r e l a t e d products discussed above. Compound 12^ and i t s o x i d a t i o n products are much weaker mutagens than a c t i v a t e d s u l f a l l a t e . The mutagenic a c t i v i t y of s u l f a l l a t e i s most e a s i l y explained by S-methylene h y d r o x y l a t i o n to give a proximate mutagen c l e a v i n g to 2-chloro­ a c r o l e i n , the u l t i m a t e mutagen and a microsomal oxidase metabo­ l i t e of s u l f a l l a t e . This hypothesis i m p l i e s , but does not depend, on the greater ease of S-CHL h y d r o x y l a t i o n of s u l f a l l a t e than of i t s thiocarbamate analog t l 2 ) . D i a l l a t e i s reported to be carcinogenic i n mice (30) and s u l f a l l a t e i n mice and r a t s (31). These h e r b i c i d e s are metaboliz­ ed to give 2 - c h l o r o a c r o l e i n , a potent mutagen. S-Methylene h y d r o x y l a t i o n may a l s o c o n t r i b u t e to the mutagenic a c t i v i t i e s of d i a l l a t e and t r i a l l a t e with 2,3-dichloro- and 2 , 3 , 3 - t r i c h l o r o a c r o l e i n s as the u l t i m a t e mutagens. These c h l o r o a c r o l e i n s may be the u l t i m a t e carcinogens as w e l l as the u l t i m a t e mutagens. Summary Ν,Ν-Dialkylthio- and dithiocarbamate h e r b i c i d e s i n c l u d e s e v e r a l S - a l k y l and S-benzyl compounds without mutagenic a c t i v i t y and three ^ - c h l o r o a l l y l d e r i v a t i v e s which are promutagens, j > - ( 2 , 3 - d i c h l o r o a l l y l ) Ν,Ν-diisopropylthiocarbamate ( d i a l l a t e ) , S - ( 2 , 3 , 3 - t r i c h l o r o a l l y l ) Ν,Ν-diisopropylthiocarbamate ( t r i a l l a t e ) and S - ( 2 - c h l o r o a l l y l ) Ν,Ν-diethyldithiocarbamate ( s u l f a l l a t e ) . D i a l l a t e and s u l f a l l a t e are a l s o carcinogens. A l a r g e number of

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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S-Chloroallyl Thio- and Dithiocarbamate Herbicides 81

products are i d e n t i f i e d from the p e r a c i d o x i d a t i o n of d i a l l a t e , t r i a l l a t e and s u l f a l l a t e . The i n i t i a l o x i d a t i o n products a r e s u l f o x i d e s with d i a l l a t e and t r i a l l a t e and a s u l f i n e with s u l ­ fallate. Sulfoxides of the S - a l k y l , S-benzyl and S - c h l o r o a l l y l thiocarbamates d i f f e r g r e a t l y i n t h e i r s t a b i l i t y , r e a c t i o n s and biological activities. The ^-benzyl and S - a l k y l s u l f o x i d e s are moderately s t a b l e a t room temperature. The l a t t e r compounds with a β-hydrogen pyrolyze a t elevated temperatures by a c i s - e l i m i n a t i o n mechanism. Under p h y s i o l o g i c a l c o n d i t i o n s they r e a d i l y carbamylate t i s s u e t h i o l s such as g l u t a t h i o n e . Sulfoxides of SÎ-(3-chloroallyl), S - ( 2 , 3 - d i c h l o r o a l l y l ) and S - ( 2 , 3 , 3 - t r i c h l o r o a l l y l ) thiocarbamates are thermally unstable and q u i c k l y undergo [2,3] sigmatropic rearrangement followed by 1,2-elimination react i o n s to y i e l d the corresponding Ν,Ν-dialkylcarbamoylsulfenyl c h l o r i d e and a c r o l e i n , 2 - c h l o r o a c r o l e i n and 2 - c h l o r o a c r y l y l c h l o r ­ ide, respectively. 2-Chloroacrolei s u l f o x i d a t i o n of d i a l l a t f a l l a t e . D i a l l a t e and t r i a l l a t e might a l s o y i e l d 2,3-dichloro a c r o l e i n and 2 , 3 , 3 - t r i c h l o r o a c r o l e i n on enzymatic ^-methylene hydroxylation. These h a l o a c r o l e i n s are very potent b a c t e r i a l mutagens. The h e r b i c i d a l a c t i v i t y of the S - a l k y l thiocarbamates i s probably associated with the carbamylating a c t i v i t i e s of t h e i r s u l f o x i d e metabolites, whereas the h e r b i c i d a l and mutagenic a c t i v i t i e s of the S - c h l o r o a l l y l t h i o - and dithiocarbamates may be due a t l e a s t i n part to the generation of potent c h l o r o a c r o l e i n mutagens and h e r b i c i d e s v i a s u l f o x i d e or S-hydroxymethylene intermediates. Acknowledgment Supported i n part by Grant 5 P01 ES 00049 from the N a t i o n a l I n s t i t u t e s of Health. Acknowledgement i s made to the ACS Petroleum Research Fund f o r p a r t i a l support of t r a v e l costs f o r I . Schuphan to p a r t i c i ­ pate i n t h i s Symposium.

Literature Cited

1. Block, E. Amer. Chem. Soc. Symp. Ser., 1981,158 , 3 . 2. Neal, R. A. Amer. Chem. Soc. Symp. Ser., 1981,158,19 . 3. Casida, J. E.; Gray, R. Α.; Tilles, H. Science, 1974, 184, 573. 4. Casida, J. E.; Kimmel, E. C.; Ohkawa, H; Ohkawa, R. Pestic. Biochem, Physiol., 1975, 5, 1. 5. Casida, J. E.; Kimmel, E. C.; Lay, M.; Ohkawa, H.; Rodebush, J. E.; Gray, R. Α.; Tseng, C. K.; Tilles, H. Environ. Quai. Safety, Suppl. 1975, Vol. 3, 675. 6. Schuphan, I.; Rosen, J. D.; Casida, J. E. Science, 1979 205, 1013.

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

SULFUR IN PESTICIDE ACTION AND METABOLISM

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7. Schuphan, I.; Casida, J. E. Tetrahedron Lett., 1979, 841. 8. Schuphan, I.; Casida, J. E. J. Agric. Food Chem., 1979, 27, 1060. 9. Tseng, C. K.; Below, J. F., J. Agric. Food Chem., 1977, 25, 1383. 10. Chen, Y. S.; Schuphan, I.; Casida, J. E. Agric. Food Chem., 1979, 27, 709. 11. Gozzo, F.; Masoero, M.; Santi, R.; Galluzzi, G.; Barton, D. H. R. Chem. & Ind., 1975, 221. 12. Mislow, K. Rec. Chem. Progr., 1967, 28, 217. 13. Bickart, P.; Carson, F. W.; Jacobus, J.; Miller, E. G.; Mislow, K. J. Am. Chem. Soc., 1968, 90, 4869. 14. Walker, F. H.; Gaughan, E. J. Deutsches Bundespatent Offenlegungsschrift, 1975, 2518544. 15. Lansbury, P. T.; Britt R W J Am Chem Soc. 1976 98 4577. 16. Segall, Y.; Schuphan, ; , , Manuscrip preparation. 17. Zwanenburg, B.; Thys, L.; Strating, J. Tetrahedron Lett., 1967, 3453. 18. Zwanenburg, B.; Kielbasinski, P. Tetrahedron, 1979, 169. 19. Chen, Y. S.; Casida, J. E. J. Agric. Food Chem., 1978, 26, 263. 20. DeBaun, J. R.; Bova, D. L.; Tseng, C. K.; Menn, J. J. J. Agric. Food Chem., 1978, 26, 1098. 21. Hubbell, J. P.; Casida, J. E. J. Agric. Food Chem., 1977, 25, 404. 22. Rosen, J. D.; Schuphan, I.; Segall, Y.; Casida, J. E. J. Agric. Food Chem., 1980, 28, 880. 23. Lay, M.-M.; Hubbell, J. P.; Casida, J. E. Science, 1975, 189, 287. 24. Lay, M.-M.; Casida, J. E. Pestic. Biochem. Physiol., 1976, 6, 442. 25. Carere, Α.; Ortali, V. Α.; Cardamone, G.; Morpurgo, G. Chem.-Biol. Interact., 1978, 22, 297. 26. Sikka, H. C.; Florczyk, P. J. Agric. Food Chem., 1978, 26, 146. 27. De Lorenzo, F.; Staiano, N.; Silengo, L.; Cortese, R. Cancer Res., 1978, 38, 13. 28. Rosen, J. D.; Segall, Y.; Casida, J. E. Mutat. Res., 1980, 78, 113. 29. Ames, Β. N.; McCann, J.; Yamasaki, E. Mutat. Res., 1975, 31, 347. 30. Innes, J. R. M. et al. J. Natl. Cancer Inst., 1969, 42, 1101. 31. "Bioassay of sulfallate for possible carcinogenicity", Natl. Cancer Inst. Carcinogenesis Tech. Rep. Ser., 115, DHEW Publ. (NIH) 78-1370, 1978. RECEIVED February 17, 1981.

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

6 Biological and Chemical Behavior of Perhalogenmethylmercapto Fungicides: Metabolism and in Vitro Reactions of Dichlofluanid in Comparison with Captan 1

I. S C H U P H A N , D. WESTPHAL , A. H A Q U E , and W. E B I N G Institut für Pflanzenschutzmittelforschung, Biologische Bundesanstalt, 1000 Berlin 33, Federal Republic of Germany

The f u n g i t o x i N-perhalogenmethylmercapt moiet was introduced f o r f u n g i c i d e s captan(1) (N-(trichloromethylthio)-Δ tetrahydrophthalimide) and f o l p e t ( 2 ) (N-(trichloromet h y l t h i o ) p h t h a l i m i d e ) contain a p e r c h l o r i n a t e d methylt h i o group (1). About a decade l a t e r s u b s t i t u t i o n of f l u o r i n e f o r one of the c h l o r i n e s i n the perchloromethylmercapto moiety l e d to chemically r e l a t e d fun­ g i c i d e s (2,3) of which d i c h l o f l u a n i d ( 3 ) ((N-fluorodichloromethylthio)-N'-N'-dimethyl-N-phenyl s u l f o n y l diamide) was commerciallized.

>-S0 -N-S-CFCl 2

2

S t r u c t u r a l modifications i n the amide moiety or the methylmercapto group of these and r e l a t e d compounds r e s u l t i n changes i n chemical and b i o l o g i c a l a c t i v i t y . Thus, the r e a c t i v i t y of the t r i c h l o r o m e t h y l d e r i v a t i ­ ves against 4-nitrothiophenol decreases i n the se­ quence 1, 2 and 4. A s i m i l a r trend holds true f o r the fluorodichloromethyl d e r i v a t i v e s 5, 6, and 3. The fluoroanalogues r e a c t 4-10 times f a s t e r with 4 - n i t r o ­ thiophenol than the t r i c h l o r o d e r i v a t i v e s as shown i n Fig. 1 (3). The f l u o r o d i c h l o r o m e t h y l t h i o f u n g i c i d e s show s i m i l a r or greater f u n g i c i d a l activity than the t r i c h l o r o m e t h y l t h i o d e r i v a t i v e s (2). This may be rel a t e d to t h e i r increased r e a c t i v i t y against b i o l o g i ­ cal thiols. 1

Current address: Max von Pettenkofer Institut, Bundesgesundheitsamt, 1000 lin 33, Federal Republic of Germany. 0097-6156/81/0158-0085$05.00/0 © 1981 American Chemical Society

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

Ber­

86

SULFUR IN PESTICIDE ACTION AND METABOLISM

δ

Ο

1(1.9»104)

2 (1.5x 104)

|TYJN-S-CFCI

2

5 (20x104)

C

>

|Tîr;N-s-CFci,

H

3

Λ 4 ( 1 . 3 » 10*)

>

CH

>N-SO,-N-S-CFCI,

6

Figure 1. Reaction rate constants K(l/mol · min) of perhalogenated methy capto derivatives in the reaction with 4-nitrothiophenol at25°C

The mode of a c t i o n of the t r i c h l o r o m e t h y l t h i o f u n g i c i d e s was studies i n Saccharomyces. Captan ex­ h i b i t e d l o s s of f u n g i t o x i c i t y i n the presence of sulfh y d r y l compounds (4;. In v i t r o r e a c t i o n products of captan with cysteine were reported to be tetrahydrophthalimide, hydrogen s u l f i d e , carbon d i s u l f i d e , t h i a z o l i d i n e - 2 - t h i o n e - 4 - c a r b o x y l i c a c i d and h y d r o c h l o r i c a c i d (4). From the r e s u l t s i t was concluded that the r e a c t i o n pathway involved thiophosgene as an unstable intermediate and i t followed that the f u n g i t o x i c pro­ p e r t i e s of captan were r e l a t e d to the trichloromethyl­ t h i o moiety. This proposal was substantiated by the f i n d i n g s that a v a r i e t y of t r i c h l o r o m e t h y l t h i o d e r i ­ v a t i v e s had f u n g i c i d a l p r o p e r t i e s (5). Reaction of f o l p e t with t h i o l s and i t s mode of a c t i o n appear to be s i m i l a r to those of captan (6). The h a l f - l i f e of captan i n water i s reported to be about 12 hours, the r e a c t i o n products being carbon d i o x i d e , h y d r o c h l o r i c a c i d and s u l f u r (7). Although captan and f o l p e t are e x t e n s i v e l y used i n a g r i c u l t u r e , only one report on captan metabolism i n animals has been published (8). Urinary metabolites of o r a l l y - a d m i n i s t r a t e d captan were i d e n t i f i e d as t h i a z o l i d i n e - 2 - t h i o n e - 4 - c a r b o x y l i c a c i d , a s a l t of dithiobis-(methanesulphonic acid) and i t s disulphide monoxide (8). There are no reports con­ cerning metabolic studies i n higher p l a n t s . Captan i s mutagenic i n b a c t e r i a l assays such as the Salmonella typhimurium "Ames" assay (9,.10,21) and

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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87

others (Λ2 Λ£). I t i s a questionable carcinogen i n mice, inducing duodenal tumors only a t extremely high (8,000-16,000 ppm) d i e t a r y l e v e l s (14) L i t t l e i s known of the b i o l o g i c a l and chemical behavior of the f l u o r o d i c h l o r o m e t h y l d e r i v a t i v e s . Riot o l y s i s of d i c h l o f l u a n i d r e s u l t s i n the formation of N,N-dimethyl-N -phenylsulphamide, phenyl isocyanate and isothiocyanates and dimethylamidosulfonyl c h l o r i d e ( . 1 5 ) · GC-MS a n a l y s i s a l s o i n d i c a t e s the presence of b i s - ( f l u o r o d i c h l o r o m e t h y l ) d i s u l f i d e and two ketones, the l a t t e r being a r t i f a c t s a r i s i n g from the solvent, acetone. D i c h l o f l u a n i d metabolism i n p l a n t s y i e l d s N,N-dimethyl-N -phenylsulphamide ( 1_6), but nothing i s 9

f

f

Figure 2.

Photolysis of dichlofluanid

known of the f a t e of the f l u o r o d i c h l o r o m e t h y l t h i o moiety. The b i o l o g i c a l and chemical behavior of the f l u o r i n e - c o n t a i n i n g d e r i v a t i v e s as compared to the t r i c h l o r o m e t h y l t h i o compounds i s of i n t e r e s t because c h l o r i n e - f l u o r i n e s u b s t i t u t i o n normally i n f l u e n c e s the behavior of organic compounds s i g n i f i c a n t l y . Thus, the formation of a f l u o r i n a t e d thiophosgene analogue intermediate might be of t o x i c o l o g i c a l importance. This r e p o r t w i l l mainly be concerned with the me­ t a b o l i c f a t e of d i c h l o f l u a n i d i n strawberries, captan i n spinach and s o i l as w e l l as i n v i t r o r e a c t i o n s of d i c h l o f l u a n i d with c e l l t h i o l s and the comparative be­ havior of metabolites i n the Ames assay.

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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Metabolism of ( f l u o r o d i c h l o r o i n Strawberries

C-methyl)Dichlofluanid

14 Spray a p p l i c a t i o n of formulated C - d i c h l o f l u a n i d (Euparen) to flowering strawberry plants i n a closed c o n t r o l l e d v e n t i l a t e d c u l t i v a t i n g system r e s u l t e d i n a recovery of 99 % radiocarbon a f t e r 36 days (Table I). TABLE I. Balance Account of C^c] D i c h l o f l u a n i d Radioa c t i v i t y a f t e r Spray A p p l i c a t i o n on Strawberry Plants under Closed Conditions R a d i o a c t i v i t y recovered from t o t a l a p p l i e d %

Sum

Extractabl Fruits

3.53 49.6

4.3 21.2

7.83 70.8

Roots

1.55

1.8

3.35

Soil

0.59

3.3

3-89

Leaves

co

5.95 0.01

2

COS Unknown volatile

0.04

Condensing water

3.59

Washing solutions

3.49

Total

98.95

The major amount of r a d i o a c t i v e m a t e r i a l (70%) was found i n leaves, 7.8% i n roots and s o i l and 6% as ' C0p« Bligh-Dyer e x t r a c t i o n of the leaves gave 49.6% of tne ^ C - l a b e l i n the chloroform and methanol-water l a y e r s while 21.2% was unextractable. F r u i t s c o n t a i ned 3.5% radiocarbon i n the chloroform and methanolwater f r a c t i o n s while 4.3% remained unextractable. A p a r a l l e l experiment i n a s i m i l a r chamber but with the top removed gave a recovery of 45% r a d i o 1

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

6. SCHUPHAN ET AL.

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Perhalogenmethylmercapto Fungicides

carbon. Leaves contained up to 36%, f r u i t s 3.1% and roots plus s o i l 5.7% of the a p p l i e d radiocarbon (Table I I ) . 1/f

TABLE I I . Balance Account of [ C] D i c h l o f l u a n i d Radioa c t i v i t y a f t e r Spray A p p l i c a t i o n on Strawberry Plants under Open Conditions R a d i o a c t i v i t y recovered from t o t a l a p p l i e d % Extractable

Sum

Unextractable

Fruits Leaves Roots Soil

32.85 0.21

0.20

36.33 0.41

1.23

4.02

5.25

Washing solutions

0.18

45.22

Total

S p e c i a l a t t e n t i o n was given to the c h a r a c t e r i z a t i o n of the r a d i o a c t i v e metabolites found i n the strawberry f r u i t s where no parent compound was detect a b l e . U n i d e n t i f i e d , very p o l a r metabolites accounted f o r 83% of the m a t e r i a l while 3% was i d e n t i f i e d as t h i a z o l i d i n e - 2 - t h i o n e - 4 - c a r b o x y l i c a c i d by two-dimens i o n a l t h i n - l a y e r chromatography. Treatment with diazomethane allowed f o r GC-MS confirmation of the l a t t e r as the methylated d e r i v a t i v e s of the t h i a z o l i d i n e . B i s - ( f l u o r o d i c h l o r o m e t h y l ) d i s u l f i d e co-chromatographed with a l a b e l e d metabolite but i n s u f f i c i e n t mat e r i a l was a v a i l a b l e f o r confirmation by mass spectrometry . Leaves contained 55% of the l a b e l as the parent compound d i c h l o f l u a n i d , 35% as very p o l a r u n i d e n t i f i e d metabolites and 10% as t h i a z o l i d i n e - 2 - t h i o n e - 4 c a r b o x y l i c a c i d . A small amount (0.2%) was b i s - ( f l u o rodichloromethyl) d i s u l f i d e as confirmed by two dimais i o n a l t i c and MS. This amount i s probably l e s s than was a c t u a l l y present as the work up procedures were not optimized to detect such a r e l a t i v e l y v o l a t i l e

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metabolite. The small amount (3.9%) of radioactivityfound i n s o i l and 3.4% found i n roots were very p o l a r and could not be characterized f u r t h e r even a f t e r dif­ f e r e n t enzymatic cleavage r e a c t i o n s or a c i d hydroly­ sis. The v o l a t i l e metabolites comprised 6% of the r a ­ d i o a c t i v i t y . Carbon dioxide formed the main component. Other compounds, i n c l u d i n g carbonyl s u l f i d e , were de­ tected only i n traces below 0.04%. S p e c i a l attempts were made to i d e n t i f y carbonyl s u l f i d e as a metabolite of d i c h l o f l u a n i d because i t would help us understand the metabolic pathway. In a previous i n v e s t i g a t i o n i n t o the metabolism of captan, CSp was found as a metabolite (4), while i n a l a t t e r study, COS was reported ( 1 2 ) V i l e s reagent (18) often used by many proved to be unsuitabl p o s s i b l pre sence of carbon d i s u l f i d e . Both COS and CS give co­ l o r e d copper chelates that can not be q u a n t i t a t i v e l y separated by t i c . Furthermore, a n a l y s i s of the mixtu­ 1

2

2^NH

+

2 9= S

_

+ Cu

2+

*

—ν

\,

b (S) re by mass spectrometry i s f u t i l e because n e i t h e r de­ r i v a t i v e gives a molecular i o n . The formation of COS was proven by passing the a i r l e a v i n g the closed plant chamber through a mixture of d i i s o p r o p y l amine and 1,1,2,3-tetrachloropropene to give the thiocarbamate, t r i a l l a t e , as a r e a c t i o n product. .CI CI

The l a t t e r was i d e n t i f i e d by GC-MS. Using t h i s tech­ nique, 0.005% of the t o t a l a p p l i e d r a d i o a c t i v i t y was shown to be COS. The low y i e l d may have been due to h y d r o l y s i s of COS to HpS and C0p. The proposed metaoolic patnway i n strawberries i s shown i n Figure 3 . Five compounds: mixed d i s u l f i d e , s u l f i d e , s u l f e n i c a c i d , thiophosgene and the GSH-rea c t i o n product have not been i d e n t i f i e d as a straw­ berry metabolite but t h e i r involvement i s very l i k e l y based on the formation of b i s - ( f l u o r o d i c h l o r o m e t h y l )

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

6.

SCHUPHAN ET AL.

:N-SO,-N-S-C-F

Perhalogenmeîhylmercapto

Fungicides

91

^N-sa-NH + R - S - S - C ; F

+ RSH

^

'

Cl

-R-S-S-R

? Hydrolysis

Dichlof luanid

^HO-S^F

Cl

-0

F^C-S-S-CrF

H-S-C?F α

cr

-HCKF)

9, CI HO-S-C-F δ *CI Λ

rJ&Y°" S

G,u

!^î_ci-*c-a(F)

^*Y>

ci

/-HCKFJ>|,H„0

#

Ο

^

'

S=*C=0

o=c=o

Figure 3.

14

Proposed metabolism of [ C] dichlofluanid in strawberries

disulfide, thiazolidine-2-thione-4-carboxylic acid and carbonyl s u l f i d e . Fluorodichloromethane sulfonic a c i d co-chromatographed with p o l a r metabolites i s o l a ­ ted from leaves. D e r i v a t i z a t i o n v i a i t s s u l f o c h l o r i d e and r e a c t i o n with cyclohexene or c y c l o h e x y l i s o n i t r i l e f a i l e d to give d e r i v a t i v e s i n s u f f i c i e n t y i e l d f o r GC-MS confirmation. Metabolism of ( t r i c h l o r o - ^ C - m e t h y l ) Captan i n S p i ­ nach and S o i l Our i n i t i a l studies on the metabolic pathway of captan i n spinach point to s i m i l a r i t i e s with d i c h l o ­ f l u a n i d metabolism i n strawberries. Preplanting t r e a t ­ ment of s o i l with ^ C - c a p t a n followed by spinach c u l ­ t i v a t i o n f o r 34 days i n a closed c o n t r o l l e d v e n t i l a ­ ted c u l t i v a t i n g system r e s u l t e d i n a recovery of 87% radiocarbon (Table I I I ) . The major amount (49%) was found i n the s o i l , 19% i n the spinach and 19% as car­ bon dioxide. Bligh-Dyer e x t r a c t i o n of the spinach ga­ ve 7.4% of the ^ C - l a b e l i n the chloroform and metha-

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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SULFUR

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METABOLISM

TABLE I I I . Balance Account of [^C] Captan R a d i o a c t i v i t y a f t e r S o i l A p p l i c a t i o n Followed by Spinach Culture under Closed Conditions R a d i o a c t i v i t y recovered from t o t a l a p p l i e d

Sum

% Extractable Spinach

7.4 30.7

Soil co

Unextractable 11.5 18.0

18.9 48.7

2

COS

0.02

Unknown volatile

0.18

Condensing water

0.07

Washing solutions

0.04

Total

87.1

nol-water l a y e r s while 11.5% was unextractable. Ext r a c t i o n of the s o i l showed that nearly a l l the ext r a c t a b l e r a d i o a c t i v i t y (30.7%) was i n the chloroform phase while 18% was unextractable. In spinach, 1.3% of the extractable r a d i o a c t i v i t y was found to be the parent compound while 3% was b i s - ( t r i c h l o r o m e t h y l ) d i s u l f i d e and 5.2% was thiazolidine-2-thione-4-carboxyl i c a c i d . At l e a s t eleven other products were present. These materials were very polar and could not be f u r ther c h a r a c t e r i z e d . Captan accounted f o r 84% of the extractable r a d i o a c t i v i t y i n s o i l . None of the polar s o i l metabolites could be i d e n t i f i e d . In V i t r o

Studies

To obtain f u r t h e r information concerning the degradation mechanism of the fluorodichloromethyl moiety,

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

6. SCHUPHAN ET AL.

Perhalogenmethyîmercapto Fungicides

93

r e a c t i o n s were c a r r i e d out between d i c h l o f l u a n i d and glutathione or cysteine. These r e a c t i o n s were p e r f o r med a t the 0.01 mmol l e v e l i n a 1:1 mixture of water/ methanol. Molar r a t i o s of d i c h l o f l u a n i d and g l u t a t h i o ne reacted immediately to form a very p o l a r d e r i v a t i ve. Time dependent t i c a n a l y s i s of the r e a c t i o n reveal e d that the radiocarbon r e t a i n e d a t the o r i g i n d i s appeared w i t h i n two hours i n the r e a c t i o n mixture but a compound i d e n t i c a l i n t i c behavior to d i c h l o f l u a n i d was formed. The odor of mercapto compounds was e v i dent. R a d i o a c t i v i t y from the o r i g i n on the t i c p l a t e s was l o s t i n open a i r w i t h i n 24 hours. Performing t h i s r e a c t i o n a t 40 C i n a closed system f i t t e d with traps f o r COS and COp absorption and passing nitrogen through i t 2-4% CoS and 18 to 22% C0« were evolve cess or glutathione of COS. The same experiments c a r r i e d out with cysteine ave 30-35% C ° o Using a two molar cysteine excess, 0 to 70% of COp were released. The remaining r a d i o a c t i v i t y i n the r e a c t i o n mixture consisted p a r t l y of t h i a z o l i d i n e - 2 - t h i o n e - 4 - c a r b o x y l i c a c i d . In a l l experiments, 5 to 10% of the r a d i o a c t i v i t y was l o s t , poss i b l y due to formation of b i s - ( f l u o r o d i c h l o r o m e t h y l ) d i s u l f i d e . This assumption i s based on odor comparison between the d i s u l f i d e and the r e a c t i o n mixture. The degradation mechanism proposed from these r e s u l t s (Figure 4) include s h o r t - l i v e d intermediates #

»-CySH

>N-SO N-S F

Ô

CI

ON-SOfNH

CyS-S-C-F CI -CyS-SCy • CySH •Dichlof l u a n i d

^ O H S N. t

s

S

ma-

ci-*6-ci(F)-H-^i 2!±£!£!i. Uc

H

-HCI(F)

KM -HCI(F)

I+

CI -

1

pic-s-s-ci CI CI — '

χ

~ ·

H0 2

s=c=o - H 2 S j*H 0

o*c=o 2

Figure 4.

14

In vitro reactions of [ C] dichlofluanid with cysteine

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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not y e t detected but necessary to understand the metab o l i c pathway. These unstable metabolites are analogous to those postulated as a r e s u l t of i n v i t r o plus i n vivo studies of captan metabolism (4,8j. In the case of d i c h l o f l u a n i d metabolism the question a r i s e s whether f l u o r i n e influences the formation of these assumed s h o r t - l i v e d intermediates. Thiophosgene espec i a l l y i s assumed to play the major part i n the mode of a c t i o n of captan. Whether thiophosgene or i t s monofluoro analogue i s involved i n the degradation pathway of d i c h l o f l u a n i d i s not c l e a r . Mutagenicity

Studies

I t i s w e l l known that captan i s a strong mutagen i n the Ames ( 9 , . 1 2 , 2 1 13) with or withou we have found that d i c h l o f l u a n i d and two of i t s metab o l i t e s , t h i a z o l i d i n e - 2 - t h i o n e - 4 - c a r b o x y l i c a c i d and b i s - ( f l u o r o d i c h l o r o m e t h y l ) d i s u l f i d e are not mutagen i c to Salmonella typhimurium TA 100. Thiophosgene i s not mutagenic when d i s s o l v e d i n dimethyl s u l f o x i d e p r i o r to t e s t i n g , but gives p o s i t i v e r e s u l t s (2 r e vertants/nmole) when tested a f t e r d i s s o l v i n g i n ethylene g l y c o l dimethyl ether or tetrahydrofuran. This i n d i c a t e s that thiophosgene i s hydrolyzed by the hydroscopic dimethyl s u l f o x i d e before i n t e r a c t i n g with the b a c t e r i a . Surprisingly, bis-(trichloromethyl) disulfide was found to be as strong a mutagen as captan, and l i k e captan and f o l p e t , d i d not need metabolic a c t i v a t i o n (Figure 5). Comparing these r e s u l t s with the negative response obtained from d i c h l o f l u a n i d and b i s - ( f l u o r o d i c h l o r o m e t h y l ) d i s u l f i d e i t can be strongl y i n f e r r e d that the f l u o r i n e atom has a fundamental i n f l u e n c e on the mutagenic a c t i v i t y of these compounds. Indeed, the t r i c h l o r o m e t h y l t h i o d e r i v a t i v e of d i c h l o f l u a n i d i s mutagenic (Figure 6) even i n the absence of microsomal a c t i v a t i o n . In contrast, compounds (5) and (6), the monofluoro analogues of captan and f o l p e t do n o t show mutagenic a c t i v i t i e s with and without metabolic a c t i v a t i o n . L i k e the other f l u o r o containing d e r i v a t i v e s they have some bactericid a l potency i n higher concentrations which i s comp l e t e l y l o s t through microsome (S-9mix) a d d i t i o n . The mutagenic potency of b i s - ( t r i c h l o r o m e t h y l ) d i s u l f i d e may be of great s i g n i f i c a n c e i n the f u r t h e r evaluation of captan. I t has been reported (190 and confirmed by us that the d i s u l f i d e i s an impurity i n t e c h n i c a l captan. Moreover, vide supre, i t was found

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

6. SCHUPHAN ET AL.

Perhalogenmethylmercapto Fungicides

95

20001 / (*S9 )

160CM

S120CH ο υ £ 800

oc 400 Φ

100 nMoles

~gÔ

Figure 5.

Mutagenic activity of captan and bis-(trichloromethyl) disulfide with S. typhimurium TA 100

140CM 8 120(H (+S9)

2100CH Φ c ο g 800

CH, r H

_

>S0 -N-S-C Cl. 2

Ô

~ βοσ

φ > φ Œ

400 200 H

20

Figure 6.

40

>heOf^-S-CFCl

60 80 nMoles

2

100

120

140

Negative response of dichlofluanid in comparison with its tr analog assayed with S. typhimurium TA 100

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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as a metabolite i n spinach a f t e r s o i l a p p l i c a t i o n of captan and i n strawberries a f t e r p l a n t a p p l i c a t i o n (20,19). Acknowledgement Supported i n part by the Ministerium f u r F o r schung und Technologie and the Deutsche Forschungsgemeinschaft. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

DBP 887 506 (17.August 1950): Standard O i l Development DBP 1193 498 (3.November 1960): Farbenfabriken Bayer A.G. K ü h l e , E.; Klauke 1964, 76, 807. Lukens, R.J.; S i s l e r , H . D . , Phytopathology, 1958, 48, 235. Lukens, R.J., In "Fungicides". E d i t o r : Torgeson, D. C., 1969, 2, 396. New York and London: Acad. Press. S i e g e l , M . R . , J. Agric. Food Chem., 1970, 18, 823. Wolfe, N.L.; Zepp, R . G . ; Doster, J.C.; Hollis, R.C., J. A g r i c . Food Chem., 1976, 24, 1041. DeBaun, J.R.; M i a u l l i s , J.B.; Knarr, J.; M i h a i l o v s k i , Α . ; Menn, J.J., Xenobiotica, 1974, 4, 101. M a r s h a l l , T.C.; Dorough, H.W.; Swim, H.E., J. A g r i c . Food Chem., 1976, 24, 560. McCann, J.; Choi, E.; Yamasaki, E.; Ames, B . N . , Proc. Nat. Acad. S c i . USA, 1975, 72, 5135. F i e s o r , G . ; Bordas, S.; Stewart, S.J., Mutation Research, 1978, 51, 151. Legator, M . S . ; K e l l y , F.J.; Green, S.; Oswald, E.J., Ann. N.Y. Acad. Sci., 1969, 160, 344. Bridges, B . A . ; Mottershead, R . P . ; Rothwell, M . A . ; Green, M . H . L . , C h e m . - B i o l . I n t e r a c t i o n s , 1972, 5, 77. National Cancer I n s t i t u t e 1977; Bioassay of cap­ tan f o r p o s s i b l e c a r c i n o g e n i c i t y , CAS No.133-06-2, NCI-CG-TR-15 Technical Report Series No. 15. C l a r k , T . ; Watkins, D . A . M . , P e s t i c . Sci., 1978, 9, 225. Vogeler, K . ; Niessen, Η . , Pflanzenschutznachr. Bayer, 1967, 20, 534. Sommers, E.; Richmond, D . V . ; P i c k a r d , J.Α., Na­ t u r e , 1967, 215, 214. V i l e s , F.J., J. Ind. Hyg. T o x . , 1940, 22, 188. Wilkes, P . S . , B u l l . Environm. Contam. T o x i c o l . , 1979, 23, 820. Westphal, D . ; Schuphan, I . ; Haque, Α . ; Ebing, W., manuscript i n p r e p a r a t i o n .

RECEIVED January 29, 1981. In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

7 Comparative Metabolism of Dithiolane Insecticides in Plants, Animals, and the Environment 1

C H I A C. KU

and I N D E R P. K A P O O R

American Cyanamid Company, Agricultural Research Division, Princeton, NJ 08540

*

(I) (Cyolane phosfolan a r e g i s t e r e d trademark of the American Cyanamid Company r e g i s t e r e d trademark o systemic οrganophosphate i n s e c t i c i d e s c o n t a i n i n g the 1,3 d i t h i o ­ lane r i n g . Phosfolan is u s e f u l f o r the c o n t r o l of s e v e r a l c o t t o n , cabbage and tobacco i n s e c t s . I t is e f f e c t i v e against many species of Spodoptera, such as the Egyptian c o t t o n leafworm,S, littoralis, I t i s a l s o e f f e c t i v e against leafhoppers, aphids, t h r i p s , mites, w h i t e f l i e s , lygus bugs, l e a f miners, cutworms, flea b e e t l e s , and alfalfa w e e v i l s , while mephosfolan i s being used f o r the c o n t r o l of many lepidopterous and other pests of c o t t o n , c o r n , r i c e , sorghum, sugarcane, e t c . , i n s e v e r a l c o u n t r i e s , e s p e c i a l l y i n the Middle East and A s i a . Among the prominent r i c e pests c o n t r o l l e d are r i c e h i s p a (Hispa armigera), r i c e g a l l midge ( P a c h y d i p l o s i s oryzae), and all major r i c e stem borers l i k e C h i l o s u p p r e s s a l i s and Tryporyza i n c e r t u l a s . The i n v e s t i g a t i o n of the metabolic f a t e s of the two C-dithiolane i n s e c t i c i d e s l a b e l e d in the imino carbon p o s i t i o n was i n i t i a t e d to study the e x c r e t i o n , t i s s u e residue behavior and the nature of the major metabolites i n r a t s . A d d i t i o n a l s t u d i e s were undertaken to determine the nature of the metabolites i n c o t t o n , rice paddy and its environment i n order to evaluate t h e i r similarities or dissimilarities w i t h the r a t m e t a b o l i t e s . T h i s information is needed by the t o x i c o l o g i s t i n determining the s i g n i f i c a n c e of the metabolites as r e s i d u e s . 14

I

II

*Denotes carbon-14 l a b e l . 1

Current address: Mobil Chemical Company, R&D, Crop Chemicals Department, P.O. Box 1028, Princeton, N J 08540.

0097-6156/81/015 8-0097$05.00/ 0 © 1981 American Chemical Society

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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Metabolism i n Rats 1. Phosfolan. When twelve male Royal Hart Wister r a t s were given a s i n g l e o r a l dose of *C-phosfolan i n peanut o i l a t l m g A g or 2 mg/kg, which i s equivalent to 10 or 20 ppm i n the feed and housed i n Delmar-Roth metabolism cages f o r c o l l e c t i o n of r e s p i r a t i o n gases and e x c r e t a , they excreted approximately 50% of the administered r a d i o a c t i v i t y and r e s p i r e d about 20% as C02 within 144 h (Table I ) . The r a d i o a c t i v e residues i n the v a r i o u s t i s s u e s were found to be d i s t r i b u t e d throughout the body w i t h lower concentrations r e s i d i n g i n f a t and muscle (Table I I ) . Chromatographic a n a l y s i s of u r i n a r y and t i s s u e r a d i o a c t i v i t y showed the presence of only one s i g n i f i c a n t m e t a b o l i t e , which was i d e n t i f i e d as thiocyanate i o n . 1


LU

•l . ι iTTVITn'^'TV'rl'hfvv^ ι

M/E

50

100

100

Ι Γ Ι Μ Μ Ι

150

ι

200

l'MHl'

250

300

(B)

N

S"

C H

2

134

co ζLU I-

5 50 LU >

LU

τ ; u'n ' 1

M/E 50

100

150

I ' » ' I ' I ' I ' I ' I ι | ' I ' I ' M

200

250

I

' r

300

Journal of Agricultural a n d Food Chemistry

Figure 5. Cl{CH ) mass spectrum of cyclic S,S-propylene dithiocarbonate (A) C imido-labeled and (B) C ethyl-labeled mephosfolan photolysates k

13

13

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

SULFUR

108 Table V I I .

IN

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ACTION

AND

METABOLISM

Recovery of mephosfolan-derived r a d i o a c t i v i t y from g o l d f i s h reared i n r i c e paddy environment i n a greenhouse. *

Exposure Time (Weeks )

Concentration (ppm) Unextracted Extractable lh

1

Total C Mephosfolan SCN i o n Unidentified

3

Total C Mephosfola SCN i o n Unidentified**

Total

1.06

1.36

0.26 0.04 Trace 0.22

0.42

0.68

0.30 0.09 0.05 0.16

lk

0.11

5

Total C Mephosfolan SCN i o n Unidentified

7

Total

1£f

C

0.20

0.57

0.77

9

Total

l H

C

0.11

0.27

0.38

1 4

* F i s h were placed i n t r e a t e d paddy water 1 week a f t e r treatment. * * C o n s i s t s of 22 spots; l a r g e s t 0.008 ppm. the s i g n i f i c a n c e o f the r e l a t i v e l y minor p e s t i c i d e uptake by the f i s h when reared i n the paddy environment. Metcalf e t . a l . (5) and Kapoor e t . a l . (6) have defined the e c o l o g i c a l m a g n i f i c a t i o n constant as the r a t i o o f the concentrat i o n o f parent compound i n the organism v s . the c o n c e n t r a t i o n of parent compound i n water. The bio-accumulation constant i s d e f i n e d as the r a t i o o f the t o t a l r a d i o a c t i v i t y c o n c e n t r a t i o n i n the organism v s . the t o t a l r a d i o a c t i v i t y c o n c e n t r a t i o n i n water. The authors obtained data f o r DDT and methoxychlor i n a t e r r e s t r i a l aquatic model ecosystem where the p e s t i c i d e was a p p l i e d t o t e r r e s t r i a l f o l i a g e only. The f i s h were kept i n t h i s system f o r three days and the highest v a l u e f o r e c o n o l o g i c a l m a g n i f i c a t i o n constant f o r DDT was 84500 and f o r bioaccumulation was 13500. The comparative f i g u r e s f o r methoxychlor, a biodegradable p e s t i c i d e , were 1545 and 206, r e s p e c t i v e l y . Although i t i s

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

7. KU AND KAPOOR

Dithiolane Insecticides

109

d i f f i c u l t to compare t h i s study w i t h those u s i n g the M e t c a l f system, due to the d i f f e r e n c e s i n the two systems, we can examine and compare t h e i r data w i t h the mephosfolan sutdy i n which f i s h were exposed f o r one week i n the paddy environment. From a s t a r t i n g c o n c e n t r a t i o n o f 0.16 ppm, an e c o l o g i c a l m a g n i f i c a t i o n of 0.56 and a bioaccumulâtion of 8.5 were found f o r f i s h exposed to mephosfolan. Since very low values of e c o l o g i c a l m a g n i f i c a t i o n (0.56 v s . 84500 f o r DDT) were obtained, i t i s evident that mephosf o l a n i s not l i k e l y to cause any e c o l o g i c a l o r bioaccumulation hazards when i t i s used i n a rice-paddy environment. Abstract 14

The metabolism of two C-dithiolaneinsecticides labeled in the imido carbon position was investigated in rats cotton plants and a simulated rice padd rats treated with C-mephosfolan by preparative and thin-layer cochromatography with synthetic standards and by mass spectroscopy showed that the attack occurs at the methyl moiety of the dithiolane ring resulting in a carboxylate ions. Hydrolysis of the P-N bond succeeded by ring opening results in release of the thiocyanate ion. This latter pathway is also the only metabolic route for phosfolan. For both insecticides, the predominant tissue metabolite was found to be the thiocyanate ion. When cotton plants were treated, mephosfolan was the predominant radioactive residue. Hydroxylation of the methyl moiety of the iminodithiolane ring and conjugation as glucoside was a significant pathway. Upon treatment of the rice paddy environment with mephosfolan, it was observed that the rice plants assimilate mephosfolan rapidly, degrade it and incorporate the C-carboninto the natural plant products. Fish kept in the paddy environment readily metabolized mephosfolan into thiocyanate and many polar metabolites. The major products of photodegradation of mephosfolan were cyclic S,S-propylene dithiocarbonate, 2-imino5-methyl-1,3-dithiolane, and diethyl phosphate. Minor products were ethyl phosphate and phosphoric acid. 14

14

Literature Cited 1. 2. 3. 4.

S i e g e l , J . R., Rosenblatt, D. H., J . Am. Chem. Soc. (1958), (80), 1753. Addor, R. W., J . H e t e r o c y l . Chem. (1970), 7, 381. W i l l i a m s , R. T., " D e t o x i f i c a t i o n Mechanisms", Wiley, New York, N.Y., (1959). Z u l a l i a n , J . , B l i n n , R. C., J . A g r i c . Food Chem. (1977), 25, 1033.

5.

M e t c a l f , R. L., Sangha, G. K., Kapoor, I. P., Environ. Sci. Technol. (1971), 5, 709.

6.

Kapoor, I. P., M e t c a l f , R. L., Hirwe, A. S., Coats, J. R. Khalsa, M. S., J . A g r i c . Food Chem. (1973), 21, 310.

RECEIVED February 19, 1981.

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

8 Roles of Iron-Sulfur Proteins in Degradation of Pesticidal Chemicals by Microorganisms F U M I O M A T S U M U R A , Κ. Y O N E Y A M A , and J. M A R S H A L L C L A R K Pesticide Research Center, Michigan State University, East Lansing, M I 48824

It is well known that in many biological systems, iron-sulfur proteins play important roles as electro transfe agent i oxidation-reductio reactions, e.g., photosynthesis carbohydrates and other organi compounds, , the iron-sulfur proteins, was isolated from an anaerobic bacterium, Clostridium pasteurianum, by Mortenson et a l . (1) in 1962, proteins of this type have been found in a wide variety of living organisms, ranging from microorganisms to higher plants and animals. The recent progress in the area of biological functions and molecular properties of iron-sulfur proteins has been phenomenal, and comprehensive summaries of these proteins have appeared in many reviews (2,3,4,5) and books (6,7,8). In this paper, the properties of some of the well understood iron-sulfur proteins will be briefly described. An effort w i l l be made then to relate these properties to their possible participation in degradation reactions on organic chemicals, and particularly on pesticide chemicals. Properties of Iron-sulfur Proteins Low Molecular Weight, Terminal Iron-Sulfur Proteins Rubredoxins. Rubredoxins are the simplest form of iron-sulfur proteins in which iron is bound to the sulfur atom of cysteine as shown in Fig. 1A. One of the first rubredoxins isolated was from an anaerobic bacterium, Clostridium pasteurianum, by Lovenberg and Sobel (9). The protein is composed of 54 amino acids and has a molecular weight of 6,000. The oxidized form has absorbance maxima at 380 and 490 nm. The biological role of the rubredoxin isolated from C . pasteurianum is still unknown. Another source of rubredoxins was found in an aerobic bacterium, Pseudomonas oleovorans, utilizing n-hexane as a carbon source (10). This particular rubredoxin differs from those commonly found in anaerobic bacteria in some of its properties: it has a molecular weight of 19,000, and one iron form of the protein is readily converted to a two-iron form (11). The rubredoxin of P. oleovorans functions as a terminal electron transfer component in an enzyme system which participates in the ωhydroxylation of fatty acids and hydrocarbons. The hydrocarbon-oxidizing 0097-6156/81/0158-011l$05.00/0 © 1981 American Chemical Society

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enzyme system is separated into three fractions: rubredoxin, N A D H rubredoxin reductase and ω-hydroxylase (10). In the presence of these three proteins, N A D H and molecular oxygen, fatty acids and n-alkanes are hydroxylated at ω-position during the electron transfer process as shown in F i g . 2 0 2 ) . A possible mechanism for hydroxylation is the formation of the alkyl hydroperoxide (R-CH^-OOH) intermediate by ω-hydroxylase. The intermediate is then reduced to the hydroxyl group by reductaserubredoxin system. Also, the ω-hydroxylase enzyme system has been shown to catalyze the epoxidation of alkenes (13). Rubredoxins have been isolated from other anaerobic bacteria such as Desulfovibrio gigas (14) and Peptostreptococcus elsdenii (15) but these rubredoxins have not been examined to the same extent as those described above. Ferredoxins. Ferredoxins are proteins which contain two or four iron atoms bound to cystein I B . There are two types o consist of two iron and two labile sulfur atoms coordinated to four cysteine residues, and bacterial type ferredoxins (bottom) consisting of four iron and four labile sulfur atoms coordinated to four cysteine residues. Plant type ferredoxins. Tagawa and Arnon (16) described the isolation of a ferredoxin from spinach chloroplast. This ferredoxin is a protein of 12,000 molecular weight, and consists of 97 amino acids (17). Spinach ferredoxin has abosrbance maxima at 325, 420 and 465 nm (18). Ferredoxins of this type have been isolated from other sources of plants and algae, e.g., alfalfa (19), taro (20), Leuceana glauca (21) and Scenedesmus (22). The proteins of thesë~îerredoxins are similar in their properties to ferredoxin from spinach. The biological functions of chloroplast ferredoxins are to mediate electron transport in the photosynthetic reaction. These ferredoxins receive electrons from light-excited chlorophyll, and reduce N A D P in the presence of ferredoxin-NADPH reductase (23). Another function of chloroplast ferredoxins is the formation o~F~ ATP in oxygen-evolving noncyclic photophosphorylation (24). With respect to the photoreduction of N A D P , it is known that microbial ferredoxins from C . pasteurianum (16) are capable of replacing the spinach ferredoxin, indicating the functional similarities of ferredoxins from completely different sources. The functions of chloroplast ferredoxins in photosynthesis and the properties of these ferredoxin proteins have been reviewed in detail by Orme-Johnson (2), Buchanan and Arnon (3), Bishop (25), and Yocum et a l . (26). —

Bacterial ferredoxins. Bacterial ferredoxin was first described in 1962 by Mortenson et al. (1) who found a low-molecular iron protein involved in electron transfer of pyruvate hydrogenase and nitrogenase in C. pasteurianum. Subsequently, a number of ferredoxins have been found in widely different types of bacteria such as photosynthetic bacteria and N^-fixing bacteria. These bacterial type ferredoxins have molecular

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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Iron-Sulfur Proteins

weights ranging from 5,000 to 10,000 as shown in Table 1. They have absorbance maxima at 390-400 nm.

Bacterial ferredoxins function primarily as electron carriers in ferredoxin-mediated oxidation reduction reactions. Some examples are: reduction of N A D , N A D P , F M N , F A D , sulfite and protons in anaerobic bacteria, CO^-fixation cycles in photosynthetic bacteria, nitrogen fixation in anaerobic nitrogen fixing bacteria, and reductive carboxylation of substrates in fermentative bacteria. The roles of bacterial ferredoxins in these reactions have been summarized by Orme-Johnson (2), Buchanan and Arnon (3), and Mortenson and Nakos (31). Iron-Sulfur Proteins as an Electron Transfer Component to Cytochrome Ρ -450

Adrenodoxin. Adrenodoxin is the only iron-sulfur protein which has been isolated from mammals adrenal cortex was purifie (32) and Omura et al. (33). It has a molecular weight of 12,638 (34) and the oxidized form of the protein shows maximal absorbances at 415 and 453 nm. Adrenodoxin acts as an electron carrier protein in the enzyme system required for steroid hydroxylation in adrenal mitochondria. In this system, electron transfer is involved with three proteins: cytochrome 450' 3oxin * flavoprotein. Reduced N A D P gives an electron to trie flavoprotein which passes the electron to adrenodoxin. Finally, reduced adrenodoxin transfers the electron to cytochrome P Q as shown in F i g . 3. The mechanism of cytochrome P c interaction with steroid, oxygen and adrenodoxin in mixed-function oxidase of adrenal cortex mitochondria has been reviewed by Estabrook et al. (35). P

a c 3 r e n o
^ Cl

Cl

Cl

H

Μ Η

> σ

δ

Ο Η

>

Ο W

Ο

m < / >

r

C/3

©

Ut

14

14

> β Figure 13. Structures of chloroform-soluble residues isolated from peanut roots treated with [Ο C] PCNB. Chloroform-soluble C accounted for 59.2% of the C in peanut roots. 14

(0.15%)

(0.04%)

CYSTEINE

HYDROXY-3-THIOPROPIONIC

ACID

Cl

Cl

(0.48%)

ACID

2

THIOACETIC

Cl

2

NH, I S-CH -CH-COOH

(3.1%)**

PENTACHLOROTHIOANISOLE

Cl

S-PENTACHLOROPHENYL-2-

2

3

S-PENTACHLOROPHENYL-2-

Cl

OH S-CH -CH-COOH

(3.9%)

UNIDENTIFIED P R O D U C T S

Cl ^

S-CH

s*

S*

s*

M

m G Χ > υ

ο

154

SULFUR

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AND

METABOLISM

Therefore, a similar pathway appears to operate in certain mammals. When S-(PCP)ThioAcetate was introduced into peanut plants, pentachlorothioanisole was not formed. However, other metabolites were detected, possibly glucose and amino acid conjugates similar to those reported for 2,4-D (17). Nonpolar methylene chloride-soluble residues. Pentachloro­ thioanisole and pentachlorothioanisole sulfoxide were present in the nonpolar methylene chloride-soluble fraction from each of the plant systems examined (Figure 14). In addition, pentachloro­ thiophenol was detected in some of these extracts. Pentachloro­ thioanisole has been reported as an important residue of PCNB in almost every biological system that has been examined for PCNB metabolism and pentachlorothiophenol has also been reported as a residue in several of these systems (6). The formation of these residues from S-(PCP)GS considered highly probable that such a system also operates in mammals in the metabolism of propachlor (jjj, ) and pentachlorothioanisole (20). In vitro studies with rat liver preparations also suggested that such a system operates in the metabolism of bromazepam (21). An in vitro enzyme system from onion was used to show that this pathway was operative in plants (£, 22). Onion was chosen as the source of enzymes because pentachlorothioanisole was an important metabolite of PCNB in onion (23). An active glutathione S-transferase system was detected in the onion enzyme system when it was assayed with [^C]PCNB and GSH (9). An initial rate of 14 nmol product/mg protein/hr was observed and a yield of 18$ was obtained in 17 hr. HPLC indi­ cated that S-(PCP)GSH was the only major conjugated product of this reaction. This was consistent with the in vivo studies with onion that showed that S-(PCP)GSH was the dominant GSH conjugate formed. In contrast, an enzyme from pea produced S-(PCP)GSH, S-(TCNP)GSH, and what appeared to be two isomeric S^-iTCPjdiGSH conjugates (60. S-(Pentachlorophenylglutathione was rapidly degraded to methylene chloride-soluble products when i t was incubated with the onion enzyme system in the absence of GSH and PCNB (Figure 17). The intial rate of this reaction, based on substrate disappearance, was 57 nmol/mg protein/hr. The primary methylene chloride-soluble product was the disulfide dimer of pentachloro­ thiophenol (9). The degradation of S-(PCP)GSH by this enzyme system was strongly inhibited by GSH, γ-glutamylglutamate, and phenylalanylglycine. The primary water-soluble component remaining after each of these reactions was shown by HPLC to be S-(PCP)GSH; however, a signficant amount of S-(PCP)-y-GluCys was detected in the reaction inhibited by γ-glutamylglutamate, and traces of this metabolite were found in most of the reaction mixtures. S-(Pentachlorophenyl)cysteine was not detected in any of the reaction mixtures. If the formation of pentachlorothio1

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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155

65i

Reaction time (hours) Pesticide B i o c h e m i s t r y and Physiology

Figure 17.

Breakdown of S-(PCP)GSH in the presence of the enzyme from onion (9)

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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phenol proceeded in the manner described in Figure 16, peptides would be expected to be inhibitors. Failure to demonstrate S-(PCP)Cys as an intermediate would not be inconsistent with this reaction sequence i f the C-S lyase reaction was much faster than the peptidase reactions. The immediate precursor of pentachlorothiophenol was assumed to be S-(PCP)Cys. Cysteine C-S lyase enzymes that convert S-aryl and S-alkyl derivatives of cysteine to pyruvate and a thioalcohol have been detected in some plant species (24, 25). When the onion enzyme system was assayed for C-S lyase activity with S-(2,4-dinitrophenyl)cysteine as the substrate, pyruvate and 2,4-dinitrothiophenol were liberated in a nearly 1:1 ratio ( £ ) . The i n i t i a l reaction was very fast (420 nmol product/mg protein/hr) and was complete within 5 min. S-(Pentachlorophenyl)cysteine was also a substrate for this enzyme system and yielded an i n i t i a l rat primary radioactive produc and mass spectrometry, was the disulfide dimer of pentachloro­ thiophenol. The cysteine C-S lyase reactions with S-(PCP)Cys and S-(2,4-dinitrophenyl)cysteine as substrates were both stimulated by GSH and γ-glutamylglutamate, peptides that inhibited the release of 2,4-dinitrothiophenol and pentachlorothiophenol dimer from their corresponding GSH conjugates. Peptide inhibition of pentachlorothiophenol formation from S-(PCP)GSH, but not from S-(PCP)Cys, is consistent with the pathway outlined in Figure 16. The immediate precursor of pentachlorothioanisole was assumed to be pentachlorothiophenol. The onion enzyme system was assayed for pentachlorothioanisole synthesis with pentachlorothiophenol and t C-methyl] S-adenosyl-methionine ([ ^C]SAM) as the substrates. An enzyme dependent reaction with an i n i t i a l rate of 22.8 nmol product/mg protein/hr was observed (9). After 1 hr the yield of pentachlorothioanisole was 13$ and after 17 hr i t was 16.5$. The reaction was stimulated by dithiothreitol and was inhibited by S-adenosyl-homocysteine. The methyl transferase reaction was easily coupled with the cysteine C-S lyase reaction by using S-[( C)PCP]Cys and nonradioactive SAM as the substrates. The rate of formation of pentachlorothioanisole from S-(PCP)Cys was the same as from pentachlorothiophenol. Large amounts of pentachlorothiophenol were formed during the coupled reaction. Apparently the methyl transferase reaction was rate-limiting in the coupled system. The direct in vitro synthesis of pentachlorothioanisole from PCNB, GSH, and ['^CjSAM in the presence of the onion enzyme system was attempted. After an initial lag period of about 30 min, a moderate rate of pentachlorothioanisole formation was observed, 2.3 nmol/mg protein/hr. After 16 hr, a yield of approximately 17$ was observed (9). It was concluded that pentachlorothioanisole detected in various plant tissues could easily be produced from the GSH pathway. Methylthioethers have not been commonly reported as metaboli14

1

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Glutathione Conjugates

157

tes of pesticides in plants; therefore, it is difficult to assess their importance as residues arising from the GSH pathway. If the postulated mechanism of pentachlorothioanisole formation in plants is correct, the formation of methylthioethers as pesti­ cide metabolites would depend upon the presence of GSH S-transferases, peptidases, cysteine C-S lyases, and methyltransferases. Methyltransferase enzymes are widespread in the plant kingdom, but they are frequently very substrate specific. The substrate specificity of the methyl transferase from the onion enzyme system was tested with 18 different substrates (Table I). Pentachlorothiophenol was the best substrate tested; however, four other substrates showed high levels of activity and only nine substrates showed less than 5% of the activity of penta­ chlorothiophenol. The three most active substrates were ortho substituted thiophenols. Summary and Conclusion The sequence of reactions that apparently occurs in the metabolism of PCNB are shown in Figure 18. The first step, con­ jugation with glutathione, occurred at several different sites. The glutathione conjugates appeared to be converted to dipeptide conjugates. In onion and corn, S-(PCP)-y-GluCys was a major residue after three days and the γ-glutamylcysteine conjugate of propachlor was an important transitory intermediate in soybean. These results were consistent with a previous obser­ vation on the metabolism of the GSH conjugate of atrazine through a γ-glutamylcysteine conjugate in sorghum (Figure 1). The cysteine conjugates appeared to be key metabolites, occupying pivotal positions in the pathway. S-(Pentachlorophenyl)cysteine was not demonstrated in vitro, but it was a minor metabolite in peanut plants. This anomoly appeared to be due to the kinetics of the various reactions. A cysteine conjugate was clearly shown to be a key intermediary metabolite in the metabolism of the GSH conjugate of atrazine in sorghum (Figure 1). The N-malonylcysteine conjugates were important metabolites of PCNB in a l l of the species except onion and the formation of S-(PCP)MalCys from S-(PCP)Cys was demonstrated in both peanut roots and in peanut cell cultures. Propachlor was also converted in high yield to an N-malonylcysteine conjugate and an N-malonyl­ cysteine conjugate of fluorodifen was reported previously (13). N-Malonylcysteine conjugates are probably common end-products in the metabolism of glutathione conjugates, perhaps analogous to the mercapturic acids produced in animals. Pentachlorothiophenol was formed in vitro from S-(PCP)Cys by a C-S lyase enzyme from onion root. This enzyme was active with S-(PCP)Cys, S-(2,4-dinitrophenyl)cysteine, and the cysteine con­ jugate of propachlor. A C-S lyase from Albizzia lophanta was previously shown to utilize a broad range of cysteine derivatives (24)·

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

Figure 18.

Metabolic pathway of PCΝΒ in higher plants

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

9.

LAMOUREUX AND RUSNESS

Table I.

159

Glutathione Conjugates

ACTIVITY OF VARIOUS SUBSTRATES FOR THE METHYL TRANSFERASE SYSTEM FROM ONION ROOT* 7

Relative Activity

Substrate 1. 2. 3. 4. 5. 6· 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Pentachlorothiophenol 2-Methoxythiopheno 2,5-Dichlorothiopheno 3-Methoxythiophenol 4-Methoxythiophenol 4-Acetamidothiophenol 4-Nitrothiophenol 2-Mercaptopyrimidine 3,4-Dichlorothiophenol 4-Methylthiophenol 4-Chlorothiophenol Thiophenol 4-Hydroxy-3-methoxycinnamic acid 4-Chlorobenzylmercaptan 2-Mercaptoacetic acid 3-Mercaptopropionic acid Pentachloroaniline Pentachlorophenol

100 4 30 20 15 11 7 6 5 4 2 2 0 0 0 0 0

The reaction mixtures contained 100 uM thiol substrate, 500 uM DTT, 100 uM [( C-methyl)]SAM, 25 mM potassium phosphate buffer (pH 7.9), and 1 mg enzyme/ml. The reaction mixtures were incubated at 30° C for 2 hr, d i luted with water, and partitioned with methylene chloride. The radioactivity in the methylene chloride phase was used as an index for the reaction. Relative activity was compared using pentachlorothiophenol as 100. All reactions were run in duplicate with appropriate controls in a manner similar to that previously described for pentachlorothiophenol (9). l4

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

160

SULFUR

IN

PESTICIDE

ACTION

AND

METABOLISM

Insoluble residue was the most abundant product of pentachlorothiophenol metabolism in peanut. The importance of this process is obviously dependent upon the presence of an active C-S lyase system. S-(Pentachlorophenyl)cysteine was also a precursor of insoluble residue in peanut, but the extent to which this involved pentachlorothiophenol as an intermediate was not determined. Additional studies are needed to determine i f insoluble residues are commonly formed from GSH conjugates by other routes. Pentachlorothioanisole was an important metabolite of PCNB in onion and barley and i t was also present in most of the other tissues. Pentachlorothiophenol was an intermediary metabolite in the in vitro conversion of PCNB to pentachlorothioanisole. The methyl transferase reaction was demonstrated with pentachlorothiophenol and a number of other substrates. The importance of this reaction evaluation. Some conversio pentachlorothioanisole sulfoxide was observed in vivo, but this reaction was not specifically investigated. S-(Pentachlorophenyl)-2-thioacetic acid and S-(PCP)ThioLactate were thought to be produced from S-(PCP)Cys by a transamination reaction. S-(Pentachlorophenyl)-2-thioacetic acid accounted for 1.3% of the in peanut roots treated with S-(PCP)Cys. S-(Pentachlorophenyl)-2-hydroxy-3-thiol-propionic acid and S-(PCP)ThioAcetate were minor metabolites and their presence was not definitively established in tissues other than peanut root. The metabolism of PCNB to S-(TCNP)GSH ultimately gave rise to S-(TCNP)MalCys in a manner that no doubt paralleled the metabolism of S-(PCP)GSH. The corresponding minor products of metabolism of S-(PCP)GSH were not observed with S-(TCNP)GSH. Several disubstituted metabolites were identified in peanut root. These were thought to be produced by a second reaction of glutathione with S-(TCNP)GSH or some metabolite derived from S-(TCNP)GSH. This would suggest that even polar compounds are not immune to conjugation with glutathione. bis-Methylmercaptotetrachlorobenzene was an important metabolite of PCNB in the Rhesus monkey (26). This metabolite might be formed from a dicysteine conjugate in the manner described for the formation of pentachlorothioanisole from S-(PCP)Cys in Figure 16 or by conversion to pentachlorothioanisole followed by a second reaction with GSH as described by Bakke et a l . (19)* The only pathway competitive with GSH conjugation, aryl nitroreduction, gave rise to pentachloroaniline. Pentachloroaniline was very slowly metabolized in peanut roots, 42% of the ^C in peanut roots was s t i l l pentachloroaniline 20 days following treatment with pentachloroaniline-UL- ^C. The major products of metabolism of pentachloroaniline appeared to be insoluble residue and products that had characteristics consistent with glutathione and N-malonylcysteine conjugates. 1

1

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

9.

LAMOUREUX AND

RUSNESS

Glutathione Conjugates

161

Evidence that pentachloroaniline may enter the GSH conjugation pathway is interesting in lieu of the fact that tetrachloroarainothioanisole was reported as a metabolite of PCNB in onion (23) and Rhesus monkey (26). These studies provided strong evidence that N-malonylcysteine conjugates may be produced from a variety of pesticide GSH conjugates in a variety of important plant species. These con­ jugates appeared to be stable end-products of metabolism. Evidence was also provided that insoluble residues may be produced from GSH conjugates via cysteine conjugate or thiol intermediates. These studies also suggested that certain reac­ tions should be studied in greater detail to assess their impor­ tance in pesticide metabolism: i . e . , the C-S lyase reaction, the methyl transferase reaction, and the transamination reaction. The possibility that the onion enzyme system might be used to classify GSH conjugate have been used to classif but there has not been a comparable method for the classification of GSH conjugates. The enzymatic conversion of S-(PCP)GSH to the dimer of pentachlorothiophenol occurred under mild conditions and was presumably specific for GSH-related peptide conjugates. The ease of isolation and stability of the onion enzyme would make it ideally suited for this purpose. Likely deficiencies in such a system would be inhibition of the peptidase reactions by contaminating peptides and failure of N-malonylcysteine conjugates to undergo the reaction. The methods used for the isolation and derivatization of the metabolites as well as the results from the detailed mass spectral studies presented in the original manuscripts (£, χ ) should have broad application in studies dealing with GSH conjugates of pesticides in plants. Glossary of Chemical Abbreviations 2,4-D DTT FAD GSH NADPH PCA PCNB PCTA PCTAS PCTP SAM S-(PCP)Cys S-(PCP)-γ-GluCys S-(PCP)GSH S-(PCP)MalCys S-(PCP)ThioAcetate

2,4-dichlorophenoxy acetic acid dithiothreitol flavin adenine dinucleotide glutathione nicotinamide adenine dinucleotide phosphate pentachloroaniline pentachloronitrobenzene pentachlorothioanisole pentachlorothioanisole sulfoxide pentachlorothiophenol S-adenosyl-L-methionine S-(pentachlorophenyl)cysteine S-(pentachlorophenyl)-γ-glutamylcysteine S-(pentachlorophenyl)glutathione S-(pentachlorophenyl)-N-malonylcysteine S-(pentachlorophenyl)-2-thioacetic acid

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

162

SULFUR

S-(PCP)ThioLactate 1

S,S -(TCP)diCys S,S·-(TCP)diGSH S-(TCNP)Cys S-(TCNP)-γ-GluCys S-(TCNP)GSH S-(TCNP)MalCys

IN

PESTICIDE

ACTION

AND

METABOLISM

S-(pentachlorophenyl)-3-thio-2-hydroxypropionic acid S,S (tetrachlorophenylene)dicysteine S,S·-(tetrachlorophenylene)diglutathione S-(tetrachloronitrophenyl)cysteine S-(tetrachloronitrophenyl)-γ-glutamylcysteine S-(tetrachloronitrophenyl)glutathione S-(tetrachloronitrophenyl)-N-malonylcysteine 1

Acknowledgment

The authors thank Jean-Marie Gouot of Rhone-Poulenc for his research on [ C]PCNB metabolism in peanut cell culture. 1I|

Abstract Pentachloronitrobenzen biochemical probe to study glutathione conjugate metabolism in plants. In peanut plants, twelve metabolites arising from glutathione conjugation of PCNB were identified. Peanut cell suspension cultures were used to study some of the precursor/ product relationships of these metabolites. Pentachloro­ nitrobenzene metabolism was also studied in soybean, cotton, corn, barley, blue green algae and onion. N-Malonylcysteine conjugates were common metabolites of PCNB in all of the species except onion. The N-Malonylcysteine conjugates of PCNB were stable in peanut cell suspension cultures for 14 days. Propachlor was also used as a biochemical probe to study gluta­ thione conjugation metabolism. Propachlor also formed an N-malonylcysteine conjugate via glutathione and γ-glutamyl­ cysteine conjugate intermediates in soybean. The N-malonyl­ cysteine conjugates appeared to be the plant kingdom equivalent to the mercapturic acids produced via glutathione conjugation in the animal kingdom. All of the species examined in this study produced significant amounts of nonextractable residue. In peanut, precursors of the nonextractable residue included S-(pentachlorophenyl)cysteine and pentachlorothiophenol. Pentachlorothioanisole was one of the metabolites formed from the metabolism of a glutathione conjugate of PCNB. The bio­ synthesis of pentachlorothioanisole from PCNB was demonstrated with an enzyme complex isolated from onion root. The enzyme complex had glutathione S-transferase, peptidase, cysteine C-S lyase, and S-methyl transferase activities. The synthesis of pentachlorothioanisole from PCNB, glutathione, and S-adenosylmethionine was catalyzed by this enzyme complex. In the absence of glutathione, the enzyme complex catalyzed the synthesis of pentachlorothioanisole from S-adenosylmethionine and either S-(pentachlorophenyl)cysteine or pentachlorothiophenol. A variety of thiophenols were tested as substrates for the methyl transferase activity. Several of these were also active.

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

9.

LAMOUREUX AND RUSNESS

Glutathione Conjugates

163

Literature Cited 1. Lamoureux, G. L.; Frear, D. S. in "Xenobiotic Metabolism: In Vitro Methods" (G. D. Paulson, D. S. Frear, and E. P. Marks, Ed.) ACS Symposium Series 97, p. 102, American Chemical Society, Washington, D.C. (1979). 2. Shimabukuro, R. H.; Lamoureux, G. L.; Frear, D. S. in "Chemistry and Action of Herbicide Antidotes" (F. M. Pallos and J. E. Casida, Ed.) p. 133, Academic Press, New York, 1978. 3. Lay, M. M.; Casida, J . E. in "Chemistry and Action of Herbicide Antidotes" (F. M. Pallos and J . E. Casida, Ed.) p. 151, Academic Press, New York, 1978. 4. Lamoureux, G. L.; Stafford Zaylskie, R. G. J . Agr

,

,

5. Lamoureux, G. L.; Frear, D. S. in "Xenobiotic Metabolism: In Vitro Methods" (G. D. Paulson, D. S. Frear, and E. P. Marks, Ed.) ACS Symposium Series 97, p. 104, American Chemical Society, Washington, D.C. (1979). 6. Lamoureux, G. L.; Rusness, D. G. J . Agr. Food Chem. 1980, 28, 1057. 7. Rusness, D. G.; Lamoureux, G. L. J . Agr. Food Chem. 1980, 28, 1070. 8. Child, J. J . ; LaRue, T. A. in "Plant Tissue Culture Methods" (O. L. Gamborg and L. R. Wetter) p. 90, National Research Council of Canada, Prairie Regional Laboratory, Saskatoon, Saskatchewan, Canada, S7N 0W9 NRCC 14383 (1975). 9. Lamoureux, G. L.; Rusness, D. G. Pest. Biochem. Physiol. 1980, 14, in press. 10. Rosa, N.; Neish, A. C. Can. J . Biochem. 1968. 46, 797. 11. Zenk, M. H.; Scherf, H. Planta 1964, 62. 12. Ladesic, B.; Pokorny, M.; Keglevic, D. P. Phytochem. 1970, 9, 2105. 13. Shimabukuro, R. H.; Walsh, W. C.; Stolzenberg, G. E.; Olson, P. A. "Abstract of Paper," Weed Sci. Soc. Amer. Meeting, Denver, Colo., Feb. 3-6, 1976.

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

SULFUR IN PESTICIDE ACTION AND METABOLISM

164

14. Shimabukuro, R. H.; Lamoureux, G. L.; Swanson, H. R.; Walsh, W. C.; Stafford, L. E.; Frear, D. S. Pest. Biochem. Physiol. 1973, 3, 483. 15. Lamoureux, G. L.; Stafford, L. E.; Tanaka, F. S. J . Agr. Food Chem. 1971, 19, 346. 16. Hubbell, J. P.; Casida, J. E. J. Agr. Food Chem. 1977, 25, 404. 17. Shimabukuro, R. H.; Lamoureux, G. L.; Frear, D. S. Pesti­ cide Metabolism in Plants: Reactions and Mechanisms, Proceedings of the US-India Seminar on Biodegredable Pesticides, April 16-19, 1979, Lucknow, India. 18. Larsen, G. L.; Bakke B14, 495. 19. Bakke, J . E.; Gustafsson, J . Α.; Gustafsson, Β. E. Science, in press. 20. Bakke, J. E.; Aschbacher, P. W.; Feil, V. J.; Gustafsson, Β. E. Xenobiotica, in press. 21. Tateishi, M.; Suzuki, S.; Shimizu, H. Biochem. Pharmacol. 1978, 27, 809. 22. Lamoureux, G. L.; Rusness, D. G. "Abstract of Paper," 178th National Meeting of the American Chemical Society, Sept., 1979. 23. Begum, S.; Scheunert, I.; Haque, Α.; Klein, W.; Korte, F. Pest. Biochem. Physiol. 1979, 11, 189. 24. Schwimmer, S.; Kjaer, A. Biochim. Biophys. Acta 1960, 42, 316. 25.

Mazelis, M.; Fowden, L. Phytochem. 1973, 12, 1287.

26. Kogel, W; Muller, W. F.; Coulston, F.; Korte, F. Chemosphere 1979, 8, 97. RECEIVED December 30, 1980.

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

10 Role of Gut Microflora in Metabolism of Glutathione Conjugates of Xenobiotics J. Ε. B A K K E , G. L. L A R S E N , and P. W. A S C H B A C H E R — M e t a b o l i s m and Radiation Research Laboratory, Agricultural Research, Science and Education Administration, U.S. Department of Agriculture, Fargo, ND 58105 J. J. R A F T E R and J. A. GUSTAFSSON—Department of Medical Nutrition, Karolinska Institute, Stockholm, Sweden Β. E. GUSTAFSSON—Department of Germfree Research, Karolinska Institute, Stockholm, Sweden

In this chapter we survey recent findings that describe the metabolic fate of acid pathway (MAP) metabolite with the bile. We show that the products of the MAP undergo an enterohepatic circulation that is mediated by intestinal en­ zymes and/or intestinal microflora. The conjugation of xenobiotics with glutathione is an important detoxication mechanism for both plants (1) and ani­ mals (2) including insects (3). In animals, these glutathione conjugates are further metabolized to mercapturic acids (S-substituted-N-acetylcysteine conjugates); in plants, the glutathione conjugates are further metabolized to S­ -substituted-N-malonylcysteine conjugates (4). In animals, evi­ dence for metabolism of a xenobiotic in the MAP has usually been the isolation of the mercapturic acid of the xenobiotic from the urine, however, the metabolic incorporation of other carbon-sulfur bonds into xenobiotics and the excretion of these metabolites as S-glucuronides in the urine (5) and/or as methylthio-, methylsulfinyl-, or methylsulfonyl-containing metabolites in the urine (6, 7) and feces (8) can also result from the catabolism of MAP metabolites. The metabolic intro­ duction o f m e t h y l t h i o - , m e t h y l s u l f i n y l - and m e t h y l s u l f o n y l -

g r o u p s i n t o x e n o b i o t i c s has been r e p o r t e d i n a t l e a s t e i g h t e e n c a s e s ( 6 , 9 - 2 5 ) , and c a f f e i n e has been shown t o be m e t a b o l i z e d to a methylthio-containing metabolite (26). C o l u c c i and B u y s k e (5) and T a t e i s h i e t a l . (27) showed t h a t r a t l i v e r c o n t a i n s enzyme s y s t e m s t h a t can p r o d u c e t h i o l s f r o m MAP m e t a b o l i t e s , and t h e l a t t e r a u t h o r s showed t h a t t h e c o r r e s p o n d i n g m e t h y l t h i o - c o n t a i n i n g m e t a b o l i t e s were f o r m e d when l i v e r m i c r o ­ somes and S - a d e n o s y l m e t h i o n i n e were added t o t h e i r c y s t e i n e conjugate β - l y a s e system. DeBaun e t a l . (9) d e s c r i b e d a pathway i n w h i c h m e t h i o n i n e s u p p l i e d t h e m e t h y l t h i o - g r o u p i n the metabolism of N - a c e t y l a m i n o f l o u r e n e . Sumio and M i o (28) have p r o p o s e d a mechanism i n w h i c h m e t h i o n i n e r e a c t e d w i t h an arene oxide t o form the m e t h y l t h i o - c o n t a i n i n g m e t a b o l i t e s from

0097-6156/81/0158-0165$05.00/0 © 1981 American Chemical Society

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

Χ =

C6H5N-C(0)CH2-

I-C3H7

X-S(0)-mercapturât^,

X-S-mercapturate

X-S-cyst

X-S-cyst-gly

X-S-glutathione

C6H5-N-C(0)CH2C1

I

i-C3H7

2)

1)

Figure 1.

intestinal enzymes and f l o r a

bile

X-S(0)-mercapt

— v \ X-S-mercapt

X-S-cyst

1 ï

flora

flora

^

X-S(0)H

/ X-SH

nonextractable fecal residues

X-S(0)2CH3

/

reabsorbed

liver

H0-X-S(0)2CH3

^

Proposed metabolic pathway for propachlor in rats

i

Enterohepatic Circulation

flora

Gl-0-X-S(0)2CH3

y

bile

10.

BAKKE ET AL.

167

Gut Microflora

2,5,2*,5,-tetrachlorobiphenyl. S t i l l w e l l e t a l . ( 2 9 ) have s u g g e s t e d mechanisms f o r m e t h y l t h i o - g r o u p i n t r o d u c t i o n i n t o t h e n a p t h a l e n e n u c l e u s based on t h e mechanism p r o p o s e d by T a t e i s h i (27) and a mechanism t h a t i n v o l v e s r e a c t i o n o f t h e e p o x i d e w i t h 2-keto-4-thiomethylbutyric acid. The p r o d u c t i o n o f m e t h y l t h i o c o n t a i n i n g m e t a b o l i t e s o f n a p t h a l e n e was d e c r e a s e d i n r a t s f e d n e o m y c i n w h i c h i n d i c a t e d an i n v o l v e m e n t o f t h e i n t e s t i n a l f l o r a in the production of these metabolites ( 2 9 ) . R e c e n t l y , we have i n j e c t e d a m e r c a p t u r i c a c i d of n a p h t h a l e n e , i s o l a t e d from u r i n e f r o m r a t s dosed w i t h ^ C - n a p h t h a l e n e , i n t o t h e cecum o f r a t s and i s o l a t e d a d i h y d r o n a p h t h a l e n e t h a t c o n t a i n e d b o t h a m e t h y l t h i o g r o u p and an O - g l u c u r o n i d e s u b s t i t u t e n t ( 3 4 ) . R e c e n t s t u d i e s f r o m o u r l a b o r a t o r i e s have shown t h e p r e s e n c e o f two pathways f o r t h e c a t a b o l i s m o f MAP m e t a b o l i t e s t h a t i n v o l v e e n t e r o h e p a t i c c i r c u l a t i o n and m i c r o f l o r a l m e t a b o ­ lism (8, 30, 31, 32). C a t a b o l i s m U t i l i z i n g M i c r o f l o r a l C-S

Lyases

P r o p a c h l o r ( 2 - c h l o r o - N - i s o p r o p y l a c e t a n i l i d e , F i g . 1) i s m e t a b o l i z e d by r a t s , p i g s and p r o b a b l y m i c e and sheep by t h e pathways o u t l i n e d i n f i g u r e 1 . In t h e f i r s t pass p r o p a c h l o r i s a b s o r b e d f r o m t h e g a s t r o i n t e s t i n a l t r a c t and q u a n t i t a t i v e l y m e t a b o l i z e d i n t h e MAP. T h i s c o n c l u s i o n i s based on t h e n e a r q u a n t i t a t i v e r e ­ c o v e r i e s o f p r o p a c h l o r d o s e s as MAP m e t a b o l i t e s ( T a b l e I ) when p r o p a c h l o r was g i v e n t o g e r m f r e e r a t s ( 3 0 ) , and b i l e d u c t c a n n u l a t e d c o n v e n t i o n a l ( 3 3 ) and g e r m f r e e r a t s ( 3 4 ) . The m a j o r b i l i a r y m e t a b o l i t e i s t h e g l u t a t h i o n e c o n j u g a t e w h i c h i s me­ t a b o l i z e d by t h e i n t e s t i n a l m i c r o f l o r a t o 2 - t h i o l o - J ^ i s o p r o p y l a c e t a n i l i d e e i t h e r d i r e c t l y or through the c y s t e i n e c o n j u g a t e as shown i n f i g u r e 2 . The c y s t e i n y l - g l y c i n e c o n ­ j u g a t e w o u l d f o l l o w t h e same p a t h w a y s . i-C3H7 Γ ' C6H5-N-C(0)CH2-S-Glutathione

C-S

lyase C6H5-N-C(0)CH2-SH

intestinal

C-S

lyase

peptidases

C6H5-N-C-CH2-S-Cysteine

Figure 2.

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

acid

mercapturic sulfoxide

a

none d e t e c t e d b number o f m e t a b o l i t e s c b i l i a r y e x c r e t i o n o f 1*C rats

68

excreted

total

C

13.0

1 4

nd

nd

66

nd

nd

4

12

nd

nd

37

68.8

nd

nd

5.7

63.1

nd

nd

nd

32.1

nd

nd

9.4

3.7

19.0

nd

nd

from separate experiments

19

nd

2 2 . 7 ( 6 )ib nd

9.5

17.6

other

methylsulfonyl containing

acid

0.0

cysteine

mercapturic

nd

nd

cysteinyl-glycine nd

nd

0.0a

27

nd

nd

7.8

19.7

0.0

0.0

0.0 0.0

0.0

67

nd

nd

6.4

5.3

4.8

nd

42.9

cannulated

65.5

nd

nd

21.8

nd

38.7

using b i l e duct

53

1.7(1)

nd

nd

5.4

1.5

14.6

28.5

C o m p a r i s o n s o f t h e m e t a b o l i s m o f p r o p a c h l o r by c o n v e n t i o n a l , g e r m f r e e and a n t i b i o t i c t r e a t e d r a t s R e c o v e r y % o f dose Conventional Germfree Antibiotic treated urine feces bi1ec urine feces b i l e c urine feces bilêc

glutathione

Table I.

10.

BAKKE ET AL.

Gut Microflora

169

I t i s p r o b a b l e t h a t t h e p e p t i d e h y d r o l y s i s r e a c t i o n s a r e accomp l i s h e d by b o t h d i g e s t i v e enzymes and m i c r o f l o r a l e n z y m e s . D i g e s t i v e enzymes a r e i m p l i c a t e d b e c a u s e t h e m a j o r m e t a b o l i t e i n g e r m f r e e r a t b i l e was t h e g l u t a t h i o n e c o n j u g a t e ( 2 8 . 3 % o f t h e dose) w h i l e t h e c y s t e i n e c o n j u g a t e was t h e m a j o r m e t a b o l i t e (19%) p r e s e n t i n g e r m f r e e r a t f e c e s ; no g l u t a t h i o n e c o n j u g a t e was p r e s e n t i n t h e g e r m f r e e f e c e s . The e x p e r i m e n t s w i t h g e r m f r e e r a t s a l s o i n d i c a t e d t h a t MAP m e t a b o l i t e s were a b l e t o undergo e n t e r o h e p a t i c c i r c u l a t i o n . The b i l e d u c t c a n n u l a t e d g e r m f r e e r a t s e x c r e t e d about 53 p e r c e n t o f o r a l doses i n t h e b i l e as MAP m e t a b o l i t e s and t h e i n t a c t g e r m f r e e r a t s e x c r e t e d o n l y 32 p e r c e n t o f s i n g l e o r a l doses i n t h e f e c e s . E n t e r o h e p a t i c c i r c u l a t i o n of the i n t a c t m e r c a p t u r i c a c i d o f p r o p a c h l o r was d e m o n s t r a t e d i n g e r m f r e e rats. G e r m f r e e r a t s g i v e n s i n g l e o r a l doses o f d u a l l a b e l e d m e r c a p t u r i c a c i d of p r o p a c h l o mercapturic acid) excrete capturic acid. Only 7.1 percent of the i s o l a t e d m e r c a p t u r i c acid contained acetate without deuterium. Preliminary results from a s i m i l a r study w i t h dual l a b e l e d c y s t e i n e conjugate ( l ^ C - p r o p a c h l o r - ^ H - c y s t e i n e ) showed t h a t i t can a l s o be a b s o r b e d f r o m t h e g a s t r o i n t e s t i n a l t r a c t and e x c r e t e d i n t h e u r i n e and b i l e as t h e d u a l l a b e l e d m e r c a p t u r i c a c i d . We have not d e t e r m i n e d i f t h e g l u t a t h i o n e , c y s t e i n y l - g l y c i n e o r , i f p r e s e n t , t h e g l u t a m y l - c y s t e i n e c o n j u g a t e s can be r e a b s o r b e d without p r i o r cleavage of peptide bonds. We have i n v i t r o e v i d e n c e t h a t t h e i n t e s t i n a l C-S l y a s e a c t i v i t y i s a s s o c i a t e d w i t h the m i c r o f l o r a . Pig cecal contents m e t a b o l i z e t h e g l u t a t h i o n e and c y s t e i n e c o n j u g a t e s and t h e m e r c a p t u r a t e t o 2 - t h i o l o - N - i s o p r o p y l a c e t a n i l i d e and n o n e x t r a c t a b l e residues. The e x t r a c t a b l e p r o d u c t o f t h e r e a c t i o n was t r a p p e d as t h e a d d u c t w i t h i o d o a c e t i c a c i d (33!) a f t e r t h e i n c u b a t i o n . W i t h o u t t h e a d d i t i o n o f i o d o a c e t i c a c i d , we were u n a b l e t o r e c o v e r any i d e n t i f i a b l e r a d i o a c t i v i t y f r o m t h e i n c u b a t e s b e c a u s e o f an a p p a r e n t r e a c t i v i t y o f t h e p r o d u c t s . The r e a c t i v i t y o f 2 - t h i o l o - N - i s o p r o p y l a c e t a n i l i d e became a p p a r e n t d u r i n g t h e s y n t h e s i s o f t h e compound. When a s o l u t i o n o f 2 - t h i o l o - N - i s o p r o p y l a c e t a n i l i d e was a l l o w e d t o s t a n d at room t e m p e r a t u r e o v e r n i g h t t h e c o r r e s p o n d i n g b i s - t h i o e t h e r was formed. Such r e a c t i v i t y c o u l d be a mechanism by w h i c h t h e n o n e x t r a c t a b l e f e c a l r e s i d u e s are formed. We have not d e t e r m i n e d where t h e _ S - m e t h y l a t i o n o f t h e C-S lyase cleavage products o c c u r s . It could take place in the f l o r a , a l t h o u g h no 2 - m e t h y l t h i o a c e t a n i l i d e s were d e t e c t e d i n t h e p i g c e c a l c o n t e n t i n c u b a t i o n s , and no m e t h y l t h i o - , o r m e t h y l s u l f o n y l - c o n t a i n i n g N - i s o p r o p y l a c e t a n i l i d e s were e x t r a c t a b l e f r o m any r a t f e c e s . The c o n v e n t i o n a l r a t f e c e s c o n t a i n e d only nonextractable residues. The m e t h y l a t i o n c o u l d a l s o t a k e p l a c e i n t h e t i s s u e s upon o r a f t e r a b s o r p t i o n o f t h e i n t e s t i n a l m e t a b o l i t e s because t h i o l S - m e t h y l t r a n s f e r a s e i s present i n

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

170

SULFUR

IN

PESTICIDE

ACTION

AND

METABOLISM

many mammalian t i s s u e s ( 3 6 ) . We c a n o n l y r e p o r t t h a t S m e t h y l a t i o n and S - o x i d a t i o n o c c u r s a t some p o i n t a f t e r t h e m i c r o f l o r a l C-S l y a s e and b e f o r e t h e b i l i a r y e x c r e t i o n o f g l u curonide conjugates of the hydroxylated 2-methylsulfonyl acetan i l i d e s i n t h e s e c o n d pass o f t h e p r o p a c h l o r m e t a b o l i t e s t h r o u g h t h e l i v e r ( 3 3 ) . The g e n e r a t i o n o f t h e s e g l u c u r o n i d e s r e s u l t s i n an e n t e r o h e p a t i c c i r c u l a t i o n o f t h e m e t h y l s u l f o n y l c o n t a i n i n g a g l y c o n e s u n t i l t h e y a r e e x c r e t e d i n t h e u r i n e as t h e a g l y c o n e s , g l u c u r o n i d e s , and o t h e r m e t a b o l i t e s o f t h e a g l y cones ( 2 0 ) . None o f t h e s e s e c o n d pass m e t a b o l i t e s were p r o duced i n g e r m f r e e r a t s o r a n t i b i o t i c t r e a t e d r a t s . The o r a l a d m i n i s t r a t i o n o f a n t i b i o t i c s r e s u l t e d i n t h e p r o d u c t i o n of germfree c h a r a c t e r i s t i c s with r e s p e c t t o p r o p a c h l o r m e t a b o l i s m i n r a t s (37) and p i g s ( 3 1 ) , i . e . no 2 - m e t h y l s u l f o n y l a c e t a n i l i d e s were formed and o n l y MAP m e t a b o l i t e s were e x c r e t e d . Thi cations. It is possibl upon i n c o r p o r a t i o n o f a n t i b i o t i c s i n t o a n i m a l f e e d c o u l d be e f f e c t e d by t h e s u p p r e s s i o n o f s u c h m e c h a n i s m s . T h i s c o u l d be a c c o m p l i s h e d e i t h e r by t h e p r e v e n t i o n o f t h e m e t a b o l i c f o r m a t i o n o f new x e n o b i o t i c s o f unknown b i o l o g i c a l a c t i v i t i e s o r by t h e c o n s e r v a t i o n o f d e t o x i c a t i o n e n e r g y o r b o t h . Species d i f f e r e n c e s i n the metabolism of propachlor are summarized i n T a b l e I I . A l l species studied metabolized p r o p a c h l o r i n t h e MAP. O b v i o u s , b u t u n e x p l a i n e d d i f f e r e n c e s a r e t h a t t h e r a t e x c r e t e d no c y s t e i n e c o n j u g a t e and t h e c h i c k e n f o r m e d no m e t h y l s u l f o n y l - c o n t a i n i n g m e t a b o l i t e s . The a b s e n c e o f m e t h y l s u l f o n y l f o r m a t i o n by c h i c k e n s i s t h o u g h t due t o t h e low b i l i a r y s e c r e t i o n o f f i r s t pass m e t a b o l i t e s . The r u m i n a n t ( s h e e p ) e x c r e t e d l a r g e amounts o f c y s t e i n e c o n j u g a t e i n u r i n e which i s a l s o not e x p l a i n e d . We do n o t know i f t h e i n t e s t i n a l f l o r a are involved i n the formation of the methylsulfonyl acet a n i l i d e s i s o l a t e d f r o m sheep u r i n e . P e n t a c h l o r o m e t h y l t h i o b e n z e n e (PCMTB) i s m e t a b o l i z e d i n rats to bis-(methylthio)tetrachlorobenzene ( b i s - M T T C B ) by a m i c r o f l o r a dependent pathway t h a t i n v o l v e s b i l i a r y e x c r e t i o n o f t h e m e r c a p t u r a t e p r e c u r s o r s and t h e a c t i o n o f a m i c r o f l o r a l C-S l y a s e ( 8 ) . The r e s u l t s o f m e t a b o l i s m s t u d i e s i n r a t s a r e outlined in table III. E i g h t y - o n e p e r c e n t of s i n g l e o r a l doses o f 1 4 C l a b e l e d PCMTB were e x c r e t e d w i t h t h e f e c e s as bis-MTTCB and n o n e x t r a c t a b l e r e s i d u e s i n about e q u a l a m o u n t s . Germfree r a t s e x c r e t e d about 88 p e r c e n t o f t h e dose w i t h t h e f e c e s as a t l e a s t two m e r c a p t u r i c a c i d s ( I , I I , f i g . 3 ) . The m a j o r m e t a b o l i t e i n t h e u r i n e f r o m b o t h c o n v e n t i o n a l and g e r m f r e e r a t s was t h e m e r c a p t u r i c a c i d .

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

D

Not

metabolites

determined

Number o f

57

Bile

a

9

22

19

Feces _.b

82

79

68

Urine

0.0

5.5(2)

13.0(3)

Other

8.8

21.7

28.3(2)

30.8

22.9

8.2(2)

9.5

11.9

13.1

22.7(6)a

acid

Mercapturic sulfoxide

17.6

0.0

9

81

0.0

0.0

16.3

48.1

15.5

£iai

23

79

0.0

11.0(2)

3

28

73

9.0(2)

0.0

12.8

34.8

7.5 0.0

28.2

Chickens

57.5

Sheep

species

R e c o v e r y % o f dose Pigs Antibiotic treated urine

Methyl s u l f o n y l containing

acid

conjugate

Mercapturic

Cysteine

Mice urine

Metabolism of p r o p a c h l o r i n v a r i o u s and a n t i b i o t i c t r e a t e d p i g s

Rat urine

Table II.

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

c

k

metabolites

B i l i a r y e x c r e t i o n of rats

Number o f

Total 1 4 C excreted a None d e t e c t e d

residues 80.7

40

nd

34

nd

nd

nd

62.8

nd

38(2)

nd

nd

nd

2.7

nd

nd

nd

nd

2.7

nd

using b i l e duct

at l e a s t 25

from s e p a r a t e experiments

5.3

4(l)b

Uncharacterized

Nonextractable

nd

nd

tetrachlorobenzene

Bismethylthio

acid

mercapturic

1

Mercapturic acid

Oxidized

nda

cannulated

88.7

nd

9.9(2)

nd

13.5

66.8

nd

Comparison of the metabolism of pentachloromethylthiobenzene i n c o n v e n t i o n a l and g e r m f r e e r a t s R e c o v e r y % of dose" Conventional Germfree feces bilec urine feces urine

Glutathione Cysteinyl-glycine Cysteine

Table I I I .

10.

BAKKE

ET

AL.

173

Gut Microflora HN-C(0)CH3 HN-C(0)CH3

CH3S-C6C14-S-CH2CHC00H I

CH3S(0)-C6Cl4-S(0)-CH2CHC00H and/or

67%

Hjl-C(0)CH3 CH3S(0)2-C6C14-S-CH2CHC00H II

13%

Figure 3. Seventy-four percen excreted i n the b i l e b 70%) has been c h a r a c t e r i z e d t o be p r o d u c t s o f t h e MAP ( 8 ) . N e i t h e r o f t h e m e r c a p t u r a t e s shown i n f i g . 3 were s e c r e t e d i n t h e b i l e t h e r e f o r e , t h e m e r c a p t u r i c a c i d s t h a t were e x c r e t e d w i t h t h e g e r m f r e e r a t f e c e s had t o have been f o r m e d e i t h e r by m e t a b o l i s m o f t h e p r e c u r s o r s o f t h e m e r c a p t u r i c a c i d by t h e i n t e s t i n a l m u c o s a , o r by t h e t i s s u e s d u r i n g e n t e r o h e p a t i c c i r c u l a t i o n of these p r e c u r s o r s . Comparison of the r a t e s of e x c r e t i o n o f o r a l doses o f PCMTB-l^C g i v e n t o g e r m f r e e and c o n v e n t i o n a l r a t s i n d i c a t e t h a t t h e r e was e n t e r o h e p a t i c c i r c u l a t i o n of the l^C i n the germfree r a t s . Conventional rats e x c r e t e d more t h a n 80 p e r c e n t o f t h e dose i n t h e f e c e s ' w i t h i n two days w h i l e i t t o o k at l e a s t e i g h t days f o r t h e g e r m f r e e r a t s t o e x c r e t e 80 p e r c e n t o f t h e dose i n t h e f e c e s . The p r e s e n c e o f n o n e x t r a c t a b l e r e s i d u e s i n t h e c o n v e n t i o n a l f e c e s i n d i c a t e s a m e t a b o l i c mechanism f o r PCMTB i n c o n v e n t i o n a l r a t s t h a t i s s i m i l a r t o t h a t f o r p r o p a c h l o r , but t h e e x c r e t i o n o f t h e bis-MTTCB w i t h t h e f e c e s i n d i c a t e s a mechanism that is different. N o t h i n g i s known about e i t h e r t h e n a t u r e o f t h e o r g a n i s m s c o n t a i n i n g t h e C-S l y a s e and t h e i r p h y s i c a l l o c a t i o n i n t h e i n t e s t i n e s , o r w h e t h e r t h e C-S l y a s e c l e a v a g e p r o d u c t i s m e t h y l a t e d by t h e f l o r a o r t h e t i s s u e s . If methylation i s a c c o m p l i s h e d by t i s s u e e n z y m e s , t h e n t h e bis-MTTCB t h a t i s e x c r e t e d w i t h t h e f e c e s must be r e s e c r e t e d i n t o t h e gut by some unknown m e c h a n i s m . B e c a u s e no b i s - M T T C B was p r e s e n t i n t h e b i l e on t h e f i r s t p a s s , i t may be p o s s i b l e t h a t t h e p r o p o s e d C-S l y a s e c l e a v a g e p r o d u c t (a m e t h y l t h i o t e t r a c h l o r o b e n z e n e t h i o l ) i s r e a b s o r b e d , m e t h y l a t e d and r e s e c r e t e d i n t h e b i l e on a second pass m e t a b o l i s m . T h i s p o s s i b i l i t y has not been i n v e s t i g a t e d as y e t . A s i m i l a r o v e r a l l mechanism c o u l d be i n v o l v e d i n the f o r m a t i o n of the m e t h y l s u l f i d e s of p o l y c h l o r i n a t e d b i p h e n y l s (PCB) t h a t a r e e x c r e t e d i n t h e f e c e s o f r a t s dosed w i t h PCBs ( 1 7 , 1 8 ) , b e c a u s e we f o u n d e v i d e n c e f o r the presence of a m e r c a p t u r i c a c i d p r e c u r s o r ( g l u t a t h i o n e c o n -

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

174

SULFUR IN PESTICIDE ACTION AND METABOLISM

j u g a t e or c y s t e i n y l - g l y c i n e conjugate) i n t h e b i l e from r a t s dosed w i t h 2 , 4 ' , 5 - t r i c h l o r o b i p h e n y l . T h e r e was no e v i d e n c e f o r any m e t h y l s u l f i d e o f t h i s PCB i n t h e b i l e , and t h e b i l e c o n t a i n e d 95 p e r c e n t o f t h e d o s e . We a l s o dosed g e r m f r e e and c o n v e n t i o n a l r a t s ( o r a l ) w i t h 2 , 4 ' , 5 - t r i c h l o r o b i p h e n y l - 1 4 c and measured t h e t i s s u e r e s i d u e l e v e l s o f 1 4 C . The s u b c u t a n e o u s and o m e n t a l f a t f r o m t h e c o n v e n t i o n a l r a t s had t i s s u e r e s i d u e s f r o m 3 t o 15 t i m e s h i g h e r t h a n t h e g e r m f r e e r a t s . A mechanism involving enterohepatic c i r c u l a t i o n could explain the pers i s t e n c e o f s u c h h a r d r e s i d u e s e s p e c i a l l y s i n c e i t i s known t h a t m e t h y l s u l f o n e s o f PCBs and DDE do a c c u m u l a t e i n t h e f a t (17).

We do n o t know w h e t h e r t h e S - o x i d i z e d m e r c a p t u r i c a c i d o f PCMTB ( I I ) i s f o r m e d i n t h e c o n v e n t i o n a l r a t o r i f i t may be a PCMTB m e t a b o l i t e t h a t i s u n i q u e t o t h e g e r m f r e e s t a t e It c o u l d r e s u l t from t i s s u c i r c u l a t i o n of the mercapturat (I). I f i t i s f o r m e d i n t h e c o n v e n t i o n a l r a t , i t may be a p r e c u r s o r f o r t h e n o n e x t r a c t a b l e f e c a l r e s i d u e s b e c a u s e no e x t r a c t a b l e m e t a b o l i t e t h a t c o n t a i n e d an o x i d i z e d s u l f u r was f o u n d i n conventional r a t excreta. As i n t h e c a s e o f p r o p a c h l o r m e r c a p t u r i c a c i d s u l f o x i d e , the b i o l o g i c a l s i g n i f i c a n c e of x e n o b i o t i c mercapturic acids t h a t c o n t a i n o x i d i z e d s u l f u r i s n o t known. C a s i d a e t a l . (39) have r e p o r t e d t h a t s u l f o x i d a t i o n o f some t h i o c a r b a m a t e h e r b i c i d e s i s a b e n e f i c i a l step i n the d e t o x i c a t i o n process. However, c y s t e i n e c o n j u g a t e s can e x h i b i t adverse b i o l o g i c a l activities. S m i t h (40) has r e v i e w e d work on t h e m e t a b o l i s m o f t h e t o x i c p r i n c i p l e i n k a l e and has shown t h a t C - S l y a s e a c t i o n on S - m e t h y l c y s t e i n e s u l f o x i d e p r o d u c e s t h e t o x i c p r i n c i p l e . V i r t a n e n (41) has r e v i e w e d t h e p r o c e s s e s i n o t h e r p l a n t s t h a t l e a d t o t h e p r o d u c t i o n o f compounds w i t h b i o l o g i c a l a c t i v i t y from S - s u b s t i t u t e d c y s t e i n e s u l f o x i d e s . C a t a b o l i s m U t i l i z i n g T i s s u e C - S L y a s e s And M i c r o f l o r a l S G l u c u r o n i d a s e s I n A d d i t i o n To The M i c r o f l o r a l C - S L y a s e To o u r k n o w l e d g e , C o l u c c i and B u y s k e (5J had t h e f i r s t r e p o r t o f MAP m e t a b o l i t e s b e i n g m e t a b o l i z e d by a h e p a t i c C - S l y a s e t h a t l e d t o t h e f o r m a t i o n o f an S - g l u c u r o n i d e . Studies on t h e m e t a b o l i s m o f 2 - a c e t a m i d o - 4 - c h l o r o m e t h y l t h i a z o l e (32) have e x t e n d e d t h i s pathway t o i n c l u d e t h e e x c r e t i o n o f t h e Sglucuronide with the b i l e . T h e s e s t u d i e s showed t h a t b o t h t h e S - g l u c u r o n i d e and t h e m e r c a p t u r i c a c i d a r e f i r s t p a s s b i l i a r y m e t a b o l i t e s o f t h i s t h i a z o l e , and t h a t b o t h o f t h e s e c o n j u g a t e s a r e m e t a b o l i z e d by m i c r o f l o r a l m e d i a t e d d e c o n j u g a t i o n pathways that u l t i m a t e l y y i e l d e d the methylthiomethyl-compounds that were e x c r e t e d i n t h e u r i n e . Our c o n c e p t i o n o f t h i s pathway i s o u t l i n e d i n f i g u r e 4 . The i n v o l v e m e n t o f t h e i n t e s t i n a l m i c r o f l o r a i n t h i s m e t a b o l i s m was shown i n t h r e e w a y s . No

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a-CH2Cl

Ν

J

CH (0)C-N-tL 3

S

H

4

X-MERCAPTURATE URINE

MERCAPTURIC ACID PATHWAY METABOLITES liver

1) 2)

bile flora

X-SH

URINE^-X-SH

I

1 i ver

1) 2)

ι

bile flora

1) 2)

X-S-GLUCURONIDE

reabsorbed metabolism

I X-SCH3 X-S(0)CH X-S(0) CH X-S-GLUCURONIDE

URINE

3

2

Ν—T-CH 2

X = CH (0)C-N-lL H

URINE

J

3

Figure 4.

3

S

Proposed metabolic pathway for 2-acetamido-4-chloromethylthiazole in rats

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

176

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4-methylthiomethyl-, 4 - m e t h y l s u l f i n y l m e t h y l - , or 4-methyls u l f o n y l m e t h y l - c o n t a i n i n g 2 - a c e t a m i d o t h i a z o l e s were p r o d u c e d by g e r m f r e e r a t s . The S - g l u c u r o n i d e and t h e m e r c a p t u r i c a c i d were i s o l a t e d f r o m t h e b i l e , and when t h e S - g l u c u r o n i d e and t h e m e r c a p t u r i c a c i d were s e p a r a t e l y i n j e c t e d i n t o t h e cecum o f r a t s , t h e m a j o r u r i n a r y m e t a b o l i t e was 2 - a c e t a m i d o - 4 - m e t h y l s u l f i n y l m e t h v l t h i a z o l e (65 and 45 p e r c e n t o f t h e d o s e s , respectively). We have no e v i d e n c e where t h e S - m e t h y l a t i o n o r oxidations occur. The e x i s t e n c e o f pathways f o r t h e i n v i v o c a t a b o l i s m o f MAP m e t a b o l i t e s t o compounds t h a t r e q u i r e f u r t h e r m e t a b o l i s m b e f o r e t h e x e n o b i o t i c m o i e t y i s e x c r e t e d f r o m t h e body i n d i ­ c a t e s t h a t m e t a b o l i s m o f x e n o b i o t i c s by t h e MAP m a y , i n some cases, represent only a transient detoxication. The t h i o l s and m e t h y l a t e d t h i o l s t h a t a r e f o r m e d r e p r e s e n t new x e n o b i o t i c s The s i g n i f i c a n c e o f t h t h e c o l o n i s n o t known showed t h a t MAP c o n j u g a t e s o f t h e 4 , 5 - e p o x i d e o f b e n z o ( a ) p y r e n e were m a j o r b i l i a r y m e t a b o l i t e s when t h e e p o x i d e was i n f u s e d i n t o the p o r t a l v e i n of r a t s . I f t h e m i c r o b i a l C-S l y a s e s y s t e m i s o p e r a t i v e i n t h e c a t a b o l i s m o f b e n z o ( a ) p y r e n e MAP m e t a b o l i t e s , t h e p r o d u c t s f o r m e d may be i n v o l v e d i n c a r ­ c i n o g e n i c p r o p e r t i e s a t t r i b u t e d t o t h e p r e s e n c e o f t h i s com­ pound i n t h e e n v i r o n m e n t . U n t i l both t h e m e t a b o l i c f a t e s of MAP m e t a b o l i t e s and S - g l u c u r o n i d e s t h a t a r e e x c r e t e d w i t h t h e b i l e and t h e b i o l o g i c a l p r o p e r t i e s o f t h e p r o d u c t s o f t h e m i c r o b i a l m e t a b o l i s m a r e known, we c a n n o t n e c e s s a r i l y c o n s i d e r t h e s e c o n j u g a t e s t o be o f no b i o l o g i c a l s i g n i f i c a n c e t o t h e host.

LITERATURE CITED

1. Shimabukuro, R. H., Lamoureux, G. L., Frear, D. S.: Eds. Pallos, F. M., Casida, J. E., "Chemistry and Action of Herbicide Antidotes"; Academic Press: New York, 1978; p. 133. 2. Chasseaud, L.F.: Eds. Arias, I. M., Jakoby, W. B., "Glutathione: Metabolism and Function"; Raven Press: New York, 1976; p. 79. 3. Brooks, G. T.: Eds. Bridges, J. W., Chasseaud, L. F., "Progress in Drug Metabolism", John Wiley and Sons: New York, 1979; Vol. 3, p. 151. 4. Lamoureux, G. L., see chapter in this publication. 5. Colucci, D. F.; Buyske, D. A. Biochem. Pharmacol., 1965, 14, 457. 6. Chatfield, D. H.; Hunter, W. H. Biochem. J., 1973, 134, 879. 7. Bakke, J. E.; Feil, V. J.; Price, C. E. Biomed. Mass Spectrom., 1976, 3, 226. 8. Bakke, J. E.; Aschbacher, P. W.; Feil, V. J.; Gustafsson, Β. E., Submitted to Xenobiotica 1980.

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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9. DeBaun, J. R.; Miller, E. C.; Miller, J. A. Cancer Res. 1970, 30, 577. 10. Focella, Α.; Heslin, P.; Teitel, S. Can. J. Chem., 1972, 50, 2025. 11. Kaul, R.; Hempel, B.; Schafer, W. Arzneim.-Forsch., 1976, 26, 489. 12. Ou, T.; Talsumi, K.; Yoshimura, H. Biochem. Biophys. Res. Commun., 1977, 75, 401. 13. Stillwell, W. G.; Bouwsma, O. J.; Thenot, J-P.; Horning, M. G. Res. Commun. Chem. Pathol. Pharmacol., 1978, 20, 509. 14. Stillwell, W. G. Pharmacologist, 1977, 19, 169. 15. Tateishi, H.; Shimizu, H. Xenobiotica, 1976, 6, 431. 16. Stock, B.; Spiteller, G. "Mass Spectrometry and Combined Techniques in Medicine Clinical Chemistry and Clinical Biochemistry", Eggstein Tubingen, F.D.R. 1977 17. Jenson, S.; Jansson, B. Ambio, 1976, 5, 257. 18. Mio, T.; Sumio, K.; Mizutani, T. Chem. Pharm. Bull., 1976, 24, 1958. 19. Miller, J. A. Cancer Res., 1970, 30, 559. 20. Bakke, J. E.; Price, C. E. J. Environ. Sci. Health, 1979, B14, 439. 21. Paulson, G. D.; Jacobsen, A. M.; Zaylskie, R. G. Pestic. Biochem. Physiol., 1977, 7, 62. 22. Kuchar, E. J . ; Geenty, F. O.; Griffith, W. P.; Thomas, R. J. J. Agr. Food Chem., 1969, 17, 1237. 23. Bray, H. G.; Hybs, Z.; James, S. P.; Thorpe, W. V. Biochem. J., 1953, 53, 266. 24. Koss, G.; Koransky, W.; Steinbach, K. Arch. Toxicol., 1979, 42, 19. 25. Walie, T. Federation Proceedings, 1977, 36, 961. 26. Kamei, K.; Matsuda, Α.; Momose, A. Chem. Pharm. Bull., 1975, 23, 683. 27. Tateishi, M.; Suzuki, S.; Shimizu, H. J. Biol. Chem., 1978, 253, 8854. 28. Sumio, K.; Mio, T. Proceedings of the First Meeting of the Japanese Society for Medical Mass Spectrometry, 1976, 1, 67. 29. Stillwell, W. G.; Bouwsma, O. J.; Horning, M. G. Res. Commun. Chem. Pathol. Pharmacol., 1978, 22, 329. 30. Bakke, J. E.; Gustafsson, J-A.; Gustafsson, Β. E. Science, 1980, 210, 433. 31. Bakke, J. E., G. L. Larsen, and P. W. Aschbacher. The Effect of Oral Lincomycin on the Metabolism of the Herbicide Propachlor by the Pig. American Society of Animal Science. 1980. (Abstract). 32. Bakke, J. E.; Feil, V. J.; Lindeskog, P.; Rafter, J. J . : Gustafsson, J-A.; Gustafsson, Β. E., submitted to Biochem. Pharmacol.

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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33. 34. 35. 36. 37. 38. 39. 40. 41. 42.

Larsen, G. L.; Bakke, J. E. The Role of Enterohepatic Circulation in the Formation of the Metabolites of 2-chloro-N-isopropylacetanilide (Propachlor), unpublished. unpublished Cole, R. D.; Stein, W. H.; Moore, S. J. Biol. Chem., 1958, 1359. Bremer, J.; Greenberg, D. M. Biochim. Biophys. Acta, 1961, 46, 217. Larsen, G. L.; Bakke, J. E. The Effect of Antibiotic Treatment on the Metabolism and Enterohepatic Circulation of Propachlor Metabolites in the Rat, unpublished. Bakke, J. E.; Price, C. E. Metabolism of 2-Chloro-NIsopropylacetanilide (Propachlor) in the Sheep and Milk Goat, J. Environ. Sci. Health 1979, B14, 291. Casida, J. E.; Gray R Α.; Tilles H Science 1964 184 573. Smith, R. H. "Report , , , 112. Virtanen, A. I. Phytochemistry 1965, 4, 207. Plummer, J. L.; Smith, B. R.; Ball, L. M.; Bend, J. R. Drug Metab. Disp., 1980, 8, 68.

RECEIVED February 5 , 1 9 8 1 .

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

INDEX Β

A A g N 0 complexation of aqueous buffer phase from washed P. putida cell incubations with Cl-toxaphene 122/ Acephate 46 2-Acetamido-4-chloromethylthiazole in rats, metabolic pathway for 175 Acyclic sulfur, compounds containin Acyclic sulfur systems 4-8 animal sources of 6-8 plant sources of 4-6 Adrenal cortex mitochondria, role of adrenodoxin in mixed-function oxidation system of 114/ Adrenodoxin 113 in mixed-function oxidation system of adrenal cortex mitochon­ dria, role of 114/ (Alkoxycarbonyl)(alkylamino)sulfenylphosphoramidothioates 46-47 S-Alkyl thiocarbamate sulfoxides, ciselimination mechanism of 68 S-Alkyl thiocarbamate sulfoxides, M C P B A oxidation of 69 Alkylsulfenyl methylcarbamates 37-39 N-Alkylsulfenyl derivatives of insecticidal methylcarbamates, toxicological properties of 37/-38/ Alkylthiosulfenylmethylcarbamates 44 Alkylthiotriazines and thiocarbamates 53-55 Allium species, organosulfur com­ pounds from 5/ Aminosulfenyl derivatives of methomyl 44 Arylsulfenyl methylcarbamates 37-39 N-Arylsulfenyl derivatives of insecticidal methylcarbamates, toxicological properties of 37/-38/ N-Arylsulfenyl derivatives of propoxur to the honey bee and house fly, toxicity of 39/ Arylsulfenylated methylcarbamates and types of selectivity 38 Arylthiosulfenylmethylcarbamates 44 Atrazine in sorghum, metabolic path­ way of 134/ 3

Bacillus megaterium degradation of mexacarbate in cellfree extracts of 124-127 effect of cofactors on the degrada­ tion of C-mexacarbate in the cell-free extracts of 126/ mixed-function oxidase system of .. 124

36

14

assays, mutageni propertie of captan in 86 Bacterial ferredoxins 112-113 molecular weight of 116/ Benzphetamine by a reconstituted monooxygenase oxidase enzyme system from rabbit liver, linearity of the metabolism of parathion and 29/ S-Benzyl thiocarbamate sulfoxides, M C P B A oxidation of 69 Bio-accumulation constant 108

C 13

C-labeled mephosfolan structures 36

106

and Cl-toxaphene by washed P. putida cells, metabolism of 121/ captan radioactivity after soil appli­ cation followed by spinach cul­ ture under closed conditions, balance account of 92/ dichlofluanid with cysteine, in vitro reactions of 93/ radioactivity after spray applica­ tion on strawberry plants under closed conditions, balance account of 88/ radioactivity after spray applica­ tion on strawberry plants under open conditions, balance account of 89/ in strawberries, proposed metabolism of 91/ -mephosfolan distribution of radioactivity in cotton plants after foliar application of 105/

181

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

182 14

SULFUR

C (continued) -mephosfolan (continued) at 2 mg/kg, excretion of radioactivity via respiration gases, urine and feces by rats treated with 101/ radioactivity levels in tissues of rats following a single oral dose of 5 mg/kg 101/ at 0.75 kg/ha, stimulated rice paddy treated with 102/ -treated paddy, residual radioactivity in rice plants grown in 105/ -mexacarbate in the cell-free extracts of B. megaterium, effect of cofactors on the degradation of 126/ -mexacarbate by ferredoxin in the presence and absence o flavin cofactor, degradatio PCNB ether-soluble metabolites isolated from the roots of peanut plants treated with 140/ formation of 80% methanolinsoluble residues in peanut plants treated with 148/ H P L C s of water-soluble extracts from peanut cell cultures treated with 144/-145/ metabolism, experimental methods 135 methylene chloride residues isolated from plant tissues treated with 152/ -treated peanut plants, from the roots of ether-soluble fractions 139-149 insoluble residue 143 methylene chloride-soluble residues 149-151 water-soluble fractions 136-139 -treated plant tissues, nonpolar methylene chloride-soluble residues from 151-157 -treated plant tissues, polar methylene chloride-soluble residues from 151-157 structures of chloroform-soluble residues isolated from peanut roots treated with 150/ water-soluble metabolites isolated from the roots of peanut plants treated with 138/ -phosfolan excretion of radioactivity via respiration gases, urine, and feces by rats treated with a single oral dose of 98/

IN

14

PESTICIDE

ACTION

AND

METABOLISM

C (continued)

-phospholan (continued) radioactivity levels in tissues of rats following a single oral dose of 2 mg/kg 99/ in rat, pathways of metabolism of 99/ in plant tissues as a function of solubility, distribution of 137/ -ring cyanatryn, elimination of radioactivity from the blood of rats dosed orally with 62/ -toxaphene by washed P. putida cells incubated aerobically, effects of added cofactors on metabolic fate of 123/ C - S lyase(s) catabolism utilizing microfloral .167-174 cleavage, 5-methylation of 169 CI(CH) cycli 5,5-propylen dithiocarbonate from C imidolabeled and C ethyl-labeled mephosfolan photolysates 107/ Cl-toxaphene, A g N 0 complexation of aqueous buffer phase from washed P. putida cell incubations with 122/ Cl-toxaphene by washed P. putida cells, metabolism of C - and .... 121/ Camphor, role of putidaredoxin in methylene hydroxylation system for 118/ Captan 85 in bacterial assays, mutagenic properties of 85 radioactivity after soil application followed by spinach culture under closed conditions, balance account of C 92/ in spinach and soil, metabolism of (trichloro- C-methyl) 91-92 and fo's-(trichloromethyl) disulfide assayed with S. typhimurium T A 100, mutagenic activity of 95/ Carbamate proinsecticides 37 Carbamates, derivatized 36 Carbofuran 40 derivatives of 43 toxicological properties of the N-sulfinyl 46/ Catabolism of glutathione conjugates of pesticides in higher plants 133-164 of M A P metabolites, pathways for the in vivo 176 utilizing microfloral C - L lyases 167-174 utilizing tissue C - S lyases and microfloral 5-glucuronidases .174-176 1 3

1 3

36

3

36

1 4

1 4

14

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

INDEX

183

S-Chloroallyl thio- and dithiocarbamate herbicides, toxicological sig­ nificance of oxidation and rear­ rangement reactions of 65-82 5-Chloroallyl thiocarbamates, Ή chemical shift data for 67/ 5-(2-Chloroallyl) N,N-diethylthiocarbamate and its oxidation prod­ ucts, Ή chemical shift data for .. 70/ 5-(3-Chloroallyl) thiocarbamate sulf­ oxides rearrangement 69 Chloroform-soluble residues isolated from peanut roots treated with C P C N B , structures of 150/ Aw-Chloroperoxybenzoic acid ( M C P B A ) , oxidation of 66 of 5-alkyl thiocarbamate sulfoxides 69 of 5-benzyl thiocarbamate sulf­ oxides 6 of 5-methallyl thiocarbamate Chloroplast ferredoxins, biologica functions of 112 Chloro-sulfallate, their sulfine deriva­ tives and further oxidation prod­ ucts, Ή chemical shift data for sulfallate 72/ Cotton plants after foliar application of C-mephosfolan, distribution of radioactivity in 105/ Cotton plants, mephosfolan metabo­ lism in 101 Cultures containing N A D H - F A D FMN 124 Cyanatryn 53, 54/ de-ethyl 56/ glutathione conjugate 57/ via liver microsomes, S-oxygenation of 59 metabolism, in vitro studies on the S-oxygenating enzyme involved in 59-60 5-oxidation of 60 5-oxide 55, 57/ and 3,4-dichlorothiophenol, reaction between 57/ and D N A , reactions between 63 in/with rat(s) elimination of radioactivity from the blood of, dosed orally with C-ring 62/ liver microsomes and N A D P H , T L C of the products derived from the incubation of C - 56/ mercapturic acids derived from the metabolism of 54/ in three species in vitro, 7V-deethylation 59/ in three species in vitro, 5-oxygenation 59/ 1 4

14

14

1 4

Cyolane phosfolan 97 Cysteine, in vitro reactions of C dichlofluanide with 93/ Cytochrome P-450 -containing monooxygenase system, metabolism of parathion to paraoxon by 23/ -containing monooxygenase system, metabolism of parathion to di­ ethyl phosphorothionate and diethyl phosphate by 25/ iron-sulfur proteins as an electron transfer component to 113-115 molecule, formation of the hydrodisulfide linkage in 32/ monooxygenase-catalyzed metabo­ lism of phosphorothionate triesters, chemical mechanisms 1 4

parathion, product Cytrolane mephosfolan

97

D De-ethyl cyanatryn 56/ N-De-ethylation of cyanatryn in three species in vitro 59/ Degradation of C-mexacarbate in the cell-free extracts of B. megaterium, effect of cofactors on 126/ of C-mexacarbate by ferredoxin in the presence and absence of flavin cofactor 119/ effects of aerobic and anaerobic culture conditions on toxaphene 120 of mexacarbate in cell-free extracts of B. megaterium 124-127 of mexacarbate by ferredoxin 117 of pesticidal chemicals by micro­ organism, roles of iron-sulfur proteins in 111-129 of toxaphene by P. putida, metabolic 117-120 Delay factor 35-49 in methylcarbamate insecticides .... 37 Desmethylmexacarbate, structures of mexacarbate and 118/ Desulfuration with p-toluenesulfonic acid 73 Dialkylaminosulfenylmethylcarbamates 43 Diallate mutagenic properties of 76 sulfoxide 74 and triallate, oxidation of 77/ 14

14

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

SULFUR

184

Dichlofluanid 85 C radioactivity after spray applica­ tion on strawberry plants under closed conditions, balance account of 88/ radioactivity after spray applica­ tion on strawberry plants under open conditions, balance account of 89/ in strawberries, proposed metab­ olism of 91/ in comparison to its trichloro analogue assayed with S. typhimuriwn T A 100, negative response of 95/ with cysteine, in vitro reactions of .. 93/ metabolism in plants 87 photolysis of 87 in strawberries, metabolism o (fluorodichloro C-methyl) 3,4-Dichlorothiophenol reaction between cyanatryn 5-oxide and .. 57/ Diethyl phosphate p-nitrophenol formation in the mammalian metabolism of parathion 24 Diethyl phosphorothionate and diethyl phosphate by the Cytochrome P-450-containing monooxygenase system, metabolism of parathion to 25/ 0,S-Dimethyl N-(N'-AlkoxycarbonyliV'-alkylaminosulfenyl)phosphoramidothioates to houseflies and mice, toxicity of 47/ N-Dimethoxyphosphinothioyl deriva­ tives of methylcarbamate esters .. 36 N,N-Dipropylthiocarbamate via Soxygenation and glutathione con­ jugation metabolism of 5-ethyl .... 58/ Disulfide, frw-(trichloromethyl) 94 Dithietane insecticide 104/ Dithietane pesticides, role of gluta­ thione conjugation in 104/ Dithiocarbamate(s) herbicides, toxicological significance of oxidation and rearrange­ ment reactions of 5-chloroallyl thio- and 65-82 metabolism of 74-75 sulfines 71-74 oxidations of 73 rearrangements 71-73 spectral features of 71 synthesis of 71 Dithiolane insecticides in plants, animals, and the environment, comparative metabolism of 97-109 D N A , reactions between cyanatryn 5-oxide and 63

IN

PESTICIDE

ACTION

AND

METABOLISM

Ε

1 4

cw-Elimination mechanism of 5-alkyl thiocarbamate sulfoxides 68 Elution profile of protein, radio­ activity, and thiocyanate from a Sephadex G-25 column of recon­ stituted monooxygenase from rat liver 31/, 32/ Enrichment in paraoxon following incubation of parathion with rabbit liver microsomes, O 21/ Enzyme involved in cyanatryn metab­ olism in vitro studies of the 5-oxygenating 59-60 Ether-soluble fractions from the roots of C PCNB-treated peanut plants 139-149 Ether-solubl metabolite isolated l s

1 4

14

Ethyl acetate phase by G L C analysis, determination of functional groups of metabolites in 125/ S-Ethyl N,N-dipropylthiocarbamate via 5-oxygenation and gluta­ thione conjugation, metabolism of 58/ F Ferredoxin(s) 112 bacterial 112-113 molecular weight of 116/ biological functions of chloroplast.. 112 degradation of mexacarbate by 117 plant-type 112 in the presence and absence of a flavin cofactor, degradation of C-mexacarbate by 119/ Fish in the rice paddy environment, metabolism in 106-109 Flavin cofactor, degradation of C mexacarbate by ferredoxin in the presence and absence of 119/ Flavin cofactor-ferredoxin system, degradation pathway of mexa­ carbate through 118/ Flavodoxin(s) as iron-sulfur protein mimics 115 isolation, microbial 115 molecular weight of selective 116/ Folpet 85 Fungicides, biological and chemical behavior of perhalogenmethylmercapto 85-96 14

1 4

5-glucuronidases, catabolism utilizing tissue C-S lyases and microfloral 174-176

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

185

INDEX

Glutathione conjugate(s) of cyanatryn 57/ of pesticides in higher plants, catabolism of 133-164 of xenobiotics, role of G U I micro­ flora in metabolism of 165-178 Glutathione conjugation in dithietane pesticides, role of 104/ in the mephosfolan metabolism in plants, role of 103 metabolism of S-ethyl Ν,Ν-άϊρτοpylthiocarbamate via S-oxygenation and 58/ of xenobiotics with 165 Goldfish reared in rice paddy environ­ ment, recovery of mephosfolanderived radioactivity from 108* G U I microflora in metabolism of glutathione conjugates of biotics, role of 165-17 H

Insecticidal methylcarbamates, toxico­ logical properties of N-alkylsulfenyl derivatives of 37/-38/ N-arylsulfenyl derivatives of 37/-38/ and the corresponding A W - t h i o biscarbamates 40/ Insecticide(s) delay factor in methylcarbamate .... 37 dithiethane 104/ Larvin—a broad-spectrum 41 in plants, animals, and the environ­ ment, comparative metabolism of dithiolane 97-109 Iron-sulfur protein(s) clusters in 114/ in degradation of pesticidal chem­ icals by microorganisms, roles of 111-129 mimics, flavodoxins as 115 in pesticide degradation, involve­ ment of 115-117 properties of 111-113 Isopropyl N-chlorosulfinyl-iV-methylcarbamate 45

Ή chemical shift data for S-chloroallyl thiocarbamates .... 67/ S-(2-chloroallyl) N,N-diethylthiocarbamate and its oxidation products 70/ L for sulfallate, chloro-sulfallate, their Larvin—a broad-spectrum insecticide 41 sulfine derivatives and further 5/ oxidation products 72/ Leukotriene C - l thiocarbamate sulfoxides 67/ Liver microsomes, the 5-oxygenation of cyanatryn via 59 Herbicide(s) Low-molecular-weight organosulfur metabolism in mammals, S-oxycompounds in nature 3-16 genation in 53-64 thiocarbamate 55 toxicological significance of oxida­ M tion and rearrangement reac­ tions of S-chloroallyl thio- and M A P (see Mercapturic acid pathway) dithiocarbamate 65-82 M C P B A (see m-Chloroperoxybenzoic Heterocyclic sulfur systems 8-10 acid) animal sources of 10 ΛΓ-Malonyl conjugates, formation of .. 143 plant sources of 8-10 iV-Malonylcysteine conjugate(s) Honey bee and house fly, toxicity of formation 139 N-arylsulfenyl derivatives of in plants 142/ propoxur to 39/ in soybean root, metabolism of House flies and mice, toxicity of 0,Spropachlor to 147/ dimethyl N-(W-alkoxycarbonylMammalian metabolism of parathion, W-alkylaminosulfenyl)-phosdiethyl phosphate p-nitrophenol phoramidothioates to 47/ formation in 24 Hydrocarbon-oxidizing enzyme system 112 Mammals, S-oxygenation in herbicide Hydrodisulfide linkage in the Cyto­ metabolism in 53-64 chrome P-450 molecule, forma­ Megaredoxin 113-115 tion of 32/ in steroid hydroxylase system of B. megaterium, role of 118/ Mephosfolan -derived radioactivity from goldfish reared in rice paddy environ­ Insecticidal methylcarbamates, Nment, recovery of 108/ alkylsulfinyl derivatives of 45

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

186

SULFUR

IN

PESTICIDE

ACTION

AND

METABOLISM

Metabolism (continued) Mephosfolan (continued) metabolism pentachloroaniline 151 of P C M T B in conventional and pathways of 100/ germfree rats, comparison of .. 172/ in plants 101-103 of phosphorothionate triesters, in cotton 101 chemical mechanisms of the in rice, study of 101 Cytochrome P-450 monooxyrole of glutathione conjugation genase-catalyzed 19-34 in 103 in rats 99-101 in plants, dichlofluanid 87 photolysates, CI(CH ) mass specin plants, mephosfolan 101-103 trum of cyclic 5,5-propylene cotton 101 dithiocarbonate from C in rice 101 imido-labeled and C ethylrole of glutathione conjugation in 103 labeled 107/ of propachlor by conventional, germfree and structures, C-labeled 106 antibiotic-treated rats, Mercapturic acid pathway ( M A P ) .... 165 metabolites, pathways for the in comparisons of 168/ species differences in 170 vivo catabolism of 176 Mercapturic acids derived from metabolism of cyanatryn in Metabolic degradation of toxaphene by C phosfolan, pathways of 99/ P. putida 117-120 mephosfolan 99-101 fate of C-toxaphene by washed phosfolan 98 P. putida cells incubated aeroof thiothiocarbamates 74-75 bically, effects of added cocarbamylation reactions of 75 factors on 123/ oxidations and rearrangements mechanism for P C M T B in rats 173 of 74-75 pathway of (trichloro- C-methyl) captan in for 2-acetamido-4-chloromethylspinach and soil 91-92 thiazole in rats 175/ in vitro studies on the 5-oxygenatof atrazine in sorghum 134/ ing enzyme involved in cyanatryn 59-60 for propachlor in rats 166/ Metabolism by washed cells, effects of added of C - and Cl-toxaphene by cofactors on toxaphene 120-124 washed P. putida cells 121/ Metabolites of C dichlofluanid in strawberries, in ethyl acetate phase by G L C proposed 91/ analysis, determination of of dithiocarbamates 74—75 functional groups of 125/ carbamylation reactions of 75 isolated from the roots of peanut oxidations and rearrangements plants treated with C P C N B , of 74-75 ether-soluble 140/ of dithiolane insecticides in plants, isolated from the roots of peanut animals, and the environment, plants treated with C P C N B , comparative 97-109 water-soluble 138/ in fish in the rice paddy environS-Methallyl thiocarbamate, M C P B A ment 106-109 oxidation of 68 of (fluorodichloro C-methyl)diMethamidophos 46 chlofluanid in strawberries 88 Methanol-insoluble residues in peanut in mammals, S-oxygenation in plants treated with C P C N B , herbicide 53-64 formation of 80% 148/ of mephosfolan, pathways of 100/ Methimazole, 5-oxygenation of 61/ of parathion Methomyl, aminosulfenyl derivatives of 44 diethyl phosphate p-nitrophenol Methyl transferase system from onion formation in the mammalian 24 root, activity of substrates for .... 159/ to paraoxon, chemical mecha5-Methylation of the C - S lyase nism of the microsomal 21 cleavage 169 products Methylcarbamates of the Cytochrome P-450 alkylsulfenyl 37-39 monooxygenase-catalyzed 24 arylsulfenyl 37-39 4

1 3

1 3

13

1 4

14

14

1 4

36

1 4

1 4

1 4

14

1 4

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

INDEX

187

Methylcarbamates (continued) and the corresponding Ν,Ν'-thiobiscarbamates, toxicological properties of insecticidal 40/ derivatives of insecticidal N-alkylsulfinyl 45 toxicological properties of . 37/-38/ iV-arylsulfinyl 45 toxicological properties of . 37/-38/ esters, N-dimethoxyphosphinothioyl 36 insecticides, delay factor in 37 and types of selectivity arylsulfenylated 38 N-Methylcarbamate, isopropyl N-chlorosulfinyl45 Methylene chloride-soluble residues from C PCNB-treated plant tissues, nonpolar 151-157 from C PCNB-treated plan tissues, polar 151-15 isolated from plant tissues with C PCNB 152/ in plants, nonpolar 154 from the roots of C PCNB-treated peanut plants 151 Methylene hydroxylation system for camphor, role of putidaredoxin in 118/ fc/s-(Methylthio)tetrachlorobenzene (to-MTTCB) 170 Mexacarbate, degradation of in cell-free extracts of B. megaterium 124-127 by ferredoxin 117 through flavin cofactor-ferredoxin system, pathway 118/ Mexacarbate and desmethylmexacarbate, structures of 118/ Mice, toxicity of 0,S-dimethyl N-(N'alkoxycarbonyl-W-alkaminosulfenyl)phosphoramidothioates to house flies 47/ Microbial flavodoxin isolation 115 Microflora in metabolism of gluta­ thione conjugates of xenobiotics, role of G U I 165-178 Microfloral C - S lyases, catabolism utilizing 167-174 Microfloral S-glucuronidases, catabo­ lism utilizing tissue C - S lyases and 174-176 Microorganism, roles of iron-sulfur proteins in degradation of pesticidal chemicals by 111-129 Microsomal metabolism of parathion to paraoxon, chemical mecha­ nism of 21 Microsomes following incubation with parathion and its metabolites, S and P bound to 26/ 1 4

Microsomes (continued) metabolism of parathion by mammalian 0 enrichment in paraoxon follow­ ing incubation of parathion with rabbit liver S-oxygenation of cyanatryn via liver Mixed-function oxidase system of

20/

1 8

B. megaterium Mixed-function oxidation system of adrenal cortex mitochondria, role of adrenodoxin in Molinate initiated by S-oxygenation, metabolism of Monooxygenase -catalyzed metabolism of parathion, products of the Cytochrome P-450

21/ 59

124 114/ 61/

24

1 4

1 4

1 4

35

3 2

ical mechanisms of the Cyto chrome P-450 19-34 oxidase enzyme system from rabbit liver, linearity of the metabo­ lism of parathion and benzphetamine by a reconstituted 29/ system metabolism of parathion to diethyl phosporothionate and diethyl phosphate by the Cytochrome P-450containing 25 metabolism of parathion to para­ oxon by the Cytochrome P-450-containing 23/ reconstituted from rabbit liver, parathion metabolism by 27/ from rat liver, elution profile of protein, radioactivity, and thiocyanate from a Sephadex G-25 column of 31/ from rat liver, S D S - P A G E of 31/ 6/5-MTTCB (fc/s-methylthio)tetrachlorobenzene) 170 Mutagenic activities of the promutagens 79/ activity of captan- and bis-(\nchloromethyl) disulfide assayed with S. typhimurium T A 100 95/ properties of captan in bacterial assays 86 of diallate 76-80 of sulfallate 80 of triallate 80 Mutagenicity studies 94

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

188

SULFUR

IN

PESTICIDE

ACTION

Ν N A D H - F A D - F M N , cultures con­ taining 124 p-Nitrophenol formation in the mam­ malian metabolism of parathion, diethyl phosphate 24 4- Nitrothiophenol at 25 °C, reaction rate constants of perhalogenated methylmercapto derivatives in the reaction with 86/ Nonpolar methylene chloride-soluble residues from C PCNB-treated plant tissues 151-157 1 4

Ο

Onion breakdown of 5-(PCP)GSH i presence of the enzyme fro enzyme system 15 assayed for pentachlorothioanisole synthesis 156 in vitro synthesis of pentachlorothioanisole from P C N B , G S H , and C S A M in the presence of 156 root, activity of substrates for the methyl transferase system from 159/ Organosulfur compounds from allium species 5/ Organosulfur compounds in nature, low-molecular-weight 3-16 Oxidation of diallate and triallate 77/ and rearrangement reactions, toxicological significance of 75-80 of 5-chloroallyl thio- and dithiocarbamate herbicides 65-82 of proherbicides 75-76 of sulfallate 78/ 5- Oxidation of cyanatryn 60 S-Oxide(s) cyanatryn 55, 57/ and D N A , reactions between ... 63 of parathion 24 reactions of 60-63 5-Oxidized mercapturic acid of PCMTB 174 S-Oxygenating enzyme involved in cyanatryn metabolism, in vitro studies on 59-60 S-Oxygenation of cyanatryn via liver microsomes 59 of cyanatryn in three sepcies in vitro 59/ in herbicide metabolism in mammals 53-64 metabolism of molinate initiated by 61/ of methimazole 61/ 1 4

AND

METABOLISM

Ρ 32

P bound to microsomes following incubation with parathion and its metabolites, S and 35

26/

P. putida (see Pseudomonas putida) P C B (polychlorinated biphenyls) P C M T B (see Pentachloromethylthiobenzene) S-(PCP) G S H , peptide inhibition of pentachlorothiophenol formation from G S H in the presence of the enzyme from onion, breakdown of thioacetate in peanut roots, origin of thiolactate in peanut roots, origin of

173

156 155/ 153f 153/

to 21 by the Cytochrome P-450-containing monooxygenase system, metabolism of parathion to .... 23/ following incubation of parathion with rabbit liver microsomes, 0 enrichment in 21/ from parathion, formation of 22 Parathion 19-34 elution from a Sephadex-25 column 30 formation of paraoxon from 22 metabolism of and benzphetamine by a reconsti­ tuted monooxygenase oxi­ dase enzyme system from rabbit liver, linearity of 29/ diethyl phosphate p-nitrophenol formation in the mammalian 24 diethyl phosphorothionate and diethyl phosphate by the Cytochrome P-450-containing monooxygenase system 25/ mammalian microsomes 20/ to paraoxon, chemical mecha­ nism of the microsomal 21 to paraoxon by the Cytochrome P-450-containing monooxy­ genase system 23/ products of 19-34 the Cytochrome P-450 mono­ oxygenase-catalyzed 24 by a reconstituted monooxy­ genase system from rabbit liver 27/ and its metabolites, S and P bound to microsomes follow­ ing incubation with 26/ S-oxides of 24 1 8

35

3 2

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

189

INDEX

Perhalogenmethylmercapto fungicides, Parathion (continued) biological and chemical behavior with rabbit liver microsomes, 0 of 85-96 enrichment in paraoxon following incubation of 2\t Perhalogenated methylmercapto derivatives in the reactions with Peanut 4-nitrothiophenol at 25°C, reaccell cultures treated with C P C N B , tion rate constants 86/ formation and stability of Pesticidal chemicals by microorganS-(PCP)MalCys and isms, roles of iron-sulfur proteins S-(TCNP)MalCys in 146/ in degradation of 111-129 cell cultures treated with C P C N B , H P L C s of water-soluble Pesticide(s) based on bioactive natural product extracts from 144/-145/ models 11/ plants treated with C P C N B , in degradation, involvement of ironthe roots of sulfur proteins in 115-117 ether-soluble fractions from 139-149 in higher plants, catabolism of ether-soluble metabolites isolated glutathione conjugates of . 1 3 3 - 1 6 4 from 140/ role of glutathione conjugation in formation of 80% methanol-insoluble residues in 148 insoluble residue in 14 types of sulfur-containing 11/ methylene chloride-soluble resi 98 dues from 149-151 Phosfolan metabolism in rats 43 water-soluble fractions from 136-139 Phosphinoaminothiomethylcarbamates Phosphorothionate triesters, chemical water-soluble metabolites mechanisms of the Cytochrome isolated 138/ P-450 monooxygenase-catalyzed roots metabolism of 19-34 origin of S-(PCP) thioacetate in 153/ Photodegradation studies 103-106 treated with C P C N B , strucPhotolysis of dichlofluanid 87/ tures chloroform-soluble residues isolated from 150/ Pigs, metabolism of propachlor in various species and antibiotic .... 171/ Pentachloroaniline metabolism 151 Plant(s) Pentachloromethylthiobenzene catabolism of glutathione conjugates (PCMTB) 170 of pesticides in higher 133-164 in conventional and germfree rats, metabolic pathway of P C N B in comparison of the metabolism higher. 158/ of 171/ metabolism in S-oxidized mercapturic acid of 174 dichlofluanid 87 in rats, metabolic mechanism for . 173 mephosfolan 101-109 Pentachloronitrobenzene (PCNB) 137/ role of glutathione conjugation in G S H , and C S A M in the presence the mephosfolan 103 of the onion enzyme system, in vitro synthesis of pentachloroN-malonylcysteine conjugates in .... 142/ nonpolar methylene chloridethioanisole from 156 soluble residues in 154 in higher plants, metabolic pathtissues as a function of solubility, way of 158/ distribution of C in 137/ Pentachlorothioanisole from P C N B , tissues, methylene chloride-soluble G S H , and C S A M in the presresidues from C PCNB-treated ence of the onion enzyme system, isolated 152/ in vitro synthesis of 156 nonpolar 151-157 Pentachlorothioanisole synthesis, polar 151-157 onion enzyme system assayed for 156 -type ferredoxins 112 Pentachlorothiophenol formation from S-(PCP) GSH, peptide inhibition Polar methylene chloride-soluble residues from C PCNB-treated of 156 plant tissues 151-157 Pentachlorothiophenol, in vitro forma173 tion of 155/ Polychlorinated biphenyls (PCB) Proherbicides, toxicological signifiPeptide inhibition of pentachlorothiocance of oxidation and rearrangephenol formation from S-(PCP) ment reactions of 75-76 GSH 156 1 8

1 4

1 4

1 4

1 4

1 4

1 4

1 4

1 4

1 4

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

SULFUR

190

IN

PESTICIDE

ACTION

AND

METABOLISM

Proinsecticides, carbamate 37 Rabbit liver (continued) parathion metabolism by a reconsti­ Promutagens, mutagenic activities of 79/ tuted monooxygenase system Propachlor 167 from 27/ metabolism (of) Radioactivity by conventional, germfree, and in cotton plants after foliar applica­ antibiotic-treated rats, tion of C-mephosfolan 105/ comparisons of 168/ from goldfish reared in rice paddy to an N-malonylcysteine conju­ environment, recovery of gate in soybean root 147/ mephosfolan-derived 108/ species differences in 170 levels in tissues of rats following a in various species and antibioticsingle oral dose of 2 mg/kg treated pigs 171/ C-phosfolan 99/ in rats 166/ levels in tissues of rats following a Propesticide action sulfur in 35-49 single oral dose of 5 mg/kg Propoxur 38 C-mephosfolan 101/ to the honey bee and house fly, via respiration gases, urine and toxicity of N-arylsulfenyl feces by rats treated with C derivatives of 39/ 5,5-Propylene dithiocarbonate fro C imido-labeled mephosfola photolysates, CI(CH ) mas spec respiratio gases, trum of cyclic 107/ feces by rats treated with a single oral dose of C-phosProteins, iron-sulfur folan, excretion of 98/ in degradation of pesticidal chem­ in rice plants grown in C-mephosicals by microorganisms, roles folan-treated paddy, residual .. 105/ of 111-129 Rat(s) as an electron transfer component comparison of the metabolism of to Cytochrome P-450 113 P C M T B in conventional and in pesticide degradation involve­ germfree 171/ ment of 115-117 comparisons of the metabolism of properties of 111-113 propachlor by conventional, Pseudomonas oleovorans, role of rugermfree, and antibioticbredoxin in ω-hydroxylation of treated 168/ alkanes of 114/ dosed orally with C-ring cyanatryn 62/ Pseudomonas putida (P. putida) cell(s), washed following a single oral dose of 2 incubated aerobically, effects of mg/kg C-phosfolan, radio­ added cofactors on metabolic activity levels in tissues of 99/ following a single oral dose of 5 fate of toxaphene by 123/ mg/kg C-mephosfolan, incubations with Cl-toxaphene, radioactivity levels in tissues of 101/ A g N 0 complexation of aqueous buffer phase from .... 122/ liver metabolic degradation of toxaphene elution profile of protein, radio­ activity, and thiocyanate by 117-120 from a Sephadex G-25 col­ metabolism of C - and Cl-toxaumn of reconstituted mono­ phene by 121/ oxygenase system from 31/, 32/ Putidaredoxin 113 microsomes and N A D P H , T L C in methylene hydroxylation systems of the products derived from for camphor, role of 118/ the incubation of C cyana­ R tryn with 56/ microsomes, sulfur bound to 30 Rabbit liver S D S - P A G E of a reconstituted linearity of the metabolism of para­ monooxygenase system from 31 / thion and benzphetamine by mephosfolan metabolism in 99-101 a reconstituted monooxygenase mercapturic acids derived from the oxidase enzyme system from .. 29/ metabolism of cyanatryn in .... 54/ microsomes, O enrichment in metabolic pathway for 2-acetamidoparaoxon following incubation 4-chloromethylthiazole in 175/ of parathion with 21/ 14

14

14

1 4

1 4

4

14

14

14

14

14

36

3

1 4

36

1 4

l s

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

191

INDEX

Sorghum, metabolic pathway of atraRat(s) (continued) zine in 134/ metabolic pathway for propachlor 166/ Soybean root, metabolism of propa­ pathways of metabolism of C chlor to an N-malonylcysteine phosfolan in 99/ conjugate in 147/ phosfolan metabolism in 98 Spinach culture under closed condi­ treated with C-mephosfolan at 2 tions, balance account of C mg/kg, excretion of radio­ captan radioactivity after soil activity via respiration gases, application followed by 92/ urine and feces by 101/ Spinach and soil, metabolism of treated with a single oral dose of (trichloro- C-methyl) captan in 91-92 C-phosfolan, excretion of Strawberry(ies) radioactivity via respiration metabolism of (fluorodichloro C gases, urine and feces by 98/ methyl) dichlofluanid in 88 Rice plants under closed conditions, environment, metabolism in fish balance account of C dichlo­ in 106-109 fluanid radioactivity after spray paddy environment, recovery in application on 88/ mephosfolan-derived radio­ activity from goldfish reare paddy treated with C-mephosfola fluanid radioactivity after spray at 0.75 kg/ha, stimulated 102/ application on 89/ plants grown in a C-mephosfolanproposed metabolism of C dichlo­ treated paddy, residual radio­ fluanid in 91/ activity in 105/ Sulfallate plants, metabolism study of chloro-sulfallate, their sulfine deriv­ mephosfolan in 101 atives and further oxidation Rubredoxin(s) 111-112 products, Ή chemical shift in ω-hydroxylation of alkanes of data for 72/ P. oleovorans, role of 114/ mutagenic properties of 80 oxidation of 78/ Sulfines, dithiocarbamate 71-74 N-Sulfinyl derivatives of carbofuran toxicological properties of 46/ S and P bound to microsomes fol­ lowing incubation with parathion N-Sulfinylmethylcarbamates 44-46 and its metabolites 26/ Sulfonamidothiomethylcarbamates . . . 43 Salmonella typhimurium (S. typhi- Sulfoxide, diallate 74 murium) 79/ Sulfoxides thiocarbamate 55, 66 T A 100 77/, 78/, 94 Sulfur mutagenic activity of captan and acyclic compounds containing 7/ 6is-(trichloromethyl disul­ bound to rat liver microsomes 30 fide assayed with 95/ -containing pesticides, some com­ negative response of dichlofluanid mon types of 11/ in comparison to its triin propesticide action 35-49 chloro analogue assayed systems, acyclic 4-8 with 95/ systems, heterocyclic 8-10 Selectivity, arylsulfenylated methyl­ carbamates and types of Τ selectivity 38 Thiaheterocycles, naturally occurring 9/ Sephadex-25 column, parathion 5/ elution from 30 Thienamycin Ν,ΛΓ-Thiobiscarbamates 40 Soil application followed by spinach toxicological properties of insecti­ culture under closed conditions, cidal methylcarbamates and balance account of C captan the corresponding 40/ radioactivity after 92/ N,W-thiobiscarbofuram 40 Soil, metabolism of (trichloro- Cmethyl) captan in spinach and 91-92 Thiocarbamate(s) Solubility, distribution of C in plant alkylthiotriazines and 53-55 tissues as a function of 137/ herbicides 55 1 4

14

1 4

14

14

1 4

1 4

14

14

1 4

35

32

1 4

14

1 4

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

192

SULFUR

Thiocarbamate(s) (continued) M C P B A oxidation of S-methallyl .. 68 sulfoxides 55, 66-77 cis-elimination mechanism of S-alkyl 68 Ή chemical shift data for 67/ M C P B A oxidation of S-alkyl 69 M C P B A oxidation of S-benzyl 69 oxidations 69 rearrangement(s) 68-69 S-(3-chloroallyl) 69 spectral features of 66-68 synthesis of 66 Ν,Ν'-Thiodicarbamates 39-43 toxicological properties of 41/ Thiothiocarbamates, metabolism of .74-75 Tissue, C - S lyases and microfloral 5-glucuronidases, catabolism utilizing 174-17 Toxaphene degradation, effects of aerobic and anaerobic culture conditions on 120 metabolism by washed cells, effects of added cofactors on 120-124 by P. putida, metabolic degradation of 117-120 Toxicity of N-arylsulfenyl derivatives of propoxur to the honey bee and house fly 39/ Toxicity of 0,S-dimethyl N-(N'alkoxycarbonyl-AT-alkylaminosulfenyl)phosphoramidothioates to house flies and mice 47/ Toxicological significance of oxidation and rearrangement reactions 75-80 balance of activation/detoxification reactions 76 of S-chloroallyl thio- and dithiocarbamate herbicides 65-82 of proherbicides 75-76 promutagens 76

IN

PESTICIDE

ACTION

AND

METABOLISM

Toxicological properties of N-alkylsulfenyl derivatives of insecticidal methylcarbamates 37/-38/ Af-arylsulfenyl derivatives of insecticidal methylcarbamates 37/-38/ of insecticidal methylcarbamates and the corresponding Ν,Ν'thiobiscarbamates 40/ the N-sulfinyl, derivatives of carbofuran 46/ Λ^'-thiodicarbamates 41/ Triallate, mutagenic properties of 80 Triallate, oxidation of diallate and .... 77/ to-CTrichloromethyl) disulfide 94 assayed with 5. typhimurium T A 100, mutagenic activity of captan and 95/

S. Typhimurium (see Salmonella

W

Water-soluble extracts from peanut cell cultures treated with C P C N B , H P L C s of 144/-145/ fractions from the roots of C PCNB-treated peanut plants 136-139 metabolites isolated from the roots of peanut plants treated with C PCNB 138/ 1 4

1 4

1 4

X

Xenobiotics with glutathione, conju­ gation of 165 Xenobiotics, role of G U I microflora in metabolism of glutathione conjugates of 165-178

In Sulfur in Pesticide Action and Metabolism; Rosen, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.