Light-Activated Pesticides (ACS Symposium Series 339)

Light-Activated Pesticides In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical S...

Author: James R. Heitz | Kelsey R. Downum

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Light-Activated Pesticides

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

ACS SYMPOSIUM SERIES

Light-Activated Pesticides James R. Heitz, E D I T O R Mississippi State University

Kelse Florida International University

Developed from a symposium sponsored by the Division of Agrochemicals at the 192nd Meeting of the American Chemical Society, Anaheim, California, September 7-12, 1986

American Chemical Society, Washington, DC 1987

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

339

Library of Congress Cataloging-in-Publication Data Light-activated pesticides. (ACS symposium series, ISSN 0097-6156; 339) American Chemical Society. Meeting (192nd: 1986: Anaheim, Calif.) Includes bibliographies and indexes. 1. Light-activated pesticides—Congresses. I. Heitz, James R., 1941. II. Downum, Kelsey R., 1952. III. American Chemical Society. Division of Agrochemicals. IV. America Meeting (192nd: 1986: Anaheim VI. Series. SB951.145.L54L54 ISBN 0-8412-1026-8

1987

668'.65

87-1342

Copyright © 1987 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each chapter in this volume indicates the copyright owner's consent that reprographic copies of the chapter 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., 27 Congress Street, Salem, MA 01970, 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 a new collective work, for resale, or for information storage and retrieval systems. The copying fee for each chapter is indicated in the code at the bottom of the first page of the chapter. 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, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected by law. PRINTED IN THE UNITED STATES OF AMERICA

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

ACS Symposium Series M . Joan Comstock, Series Editor 1987 Advisory Board H a r v e y W. B l a n c h University of California—Berkeley

V i n c e n t D. M c G i n n i s s Battelle Columbus Laboratories

Alan Elzerman Clemson University

W. H . N o r t o n

John W. F i n l e y Nabisco Brands, Inc.

James C . R a n d a l l Exxon Chemical Company

M a r y e A n n e Fox The University of Texas—Austin

E. Reichmanis AT&T Bell Laboratories

Martin L . Gorbaty Exxon Research and Engineering Co.

C. M . Roland U.S. Naval Research Laboratory

R o l a n d F. H i r s c h U.S. Department of Energy

W. D. Shults Oak Ridge National Laboratory

G . Wayne Ivie USDA, Agricultural Research Service

Geoffrey K . S m i t h Rohm & Haas Co.

R u d o l p h J. M a r c u s Consultant, Computers & Chemistry Research

D o u g l a s B. Walters National Institute of Environmental Health

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Foreword The ACS S Y M P O S I U M S E R I E S was founded in 1974 to provide a medium for publishing symposia quickly in book form. The format of the Series parallels that of the continuing A D V A N C E S IN C H E M I S T R Y S E R I E S 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 lished papers are not accepted. Both reviews and reports of research are acceptable, because symposia may embrace both types of presentation.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Preface WORLD A G R I C U L T U R A L N E E D S H A V E E X P A N D E D as world population has expanded. The pressures on agricultural productivity caused by pests (e.g., insects, weeds, and fungi) are becoming critical. At the same time, deregistration of pesticides because of safety considerations and loss of the efficacy of pesticides because of resistance threaten existing control methods. Although the catalytic action of light on the toxicity of certain chemicals in biological system exploitation of this mechanism as a watershed for new pesticides began in earnest around 1970. Since then, a rapidly increasing interest in the approach has led to the development of compounds active against agricultural pests. The first patents were issued recently, and commercial products were registered. At the same time, scientists working somewhat independently of one another in such diverse fields as synthetic dyes, natural products, and chemical intermediates that lead to photodynamically active chlorophyll derivatives were building research programs. The symposium from which this book was developed was originally intended to be a forum in which these scientists could meet and discuss their results, cross-fertilize ideas, and enlighten those not comfortably conversant with light-activated pesticides. The book grew out of the fact that no single comprehensive treatment of light-activated pesticides existed, although portions of the topic had been treated elsewhere. We would like this volume to serve as a single source for anyone interested in obtaining state-of-the-art knowledge of light-activated pesticides as well as the fundamental principles upon which the topic is built. Comprehensive chapters should enable any interested scientist to develop a complete library of the original literature upon which the chapters are based. We hope that this book becomes a "bible" for anyone interested in light-activated pesticides. We thank Monsanto Agricultural Product Company and FMC Corporation for their generous financial support of the symposium and the

ix

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Division of Agrochemicals of the American. Chemical Society for sponsoring the forum. We also thank the authors for providing quality chapters in a professional and timely manner. Finally, the quality of any book depends to some extent on the quality of anonymous reviews. We thank the reviewers whose invaluable suggestions strengthened the individual chapters. JAMES R. HEITZ

Department of Biochemistry Mississippi State University Mississippi State, MS 39762 KELSEY

R.

DOWNUM

Department of Biological Sciences Florida International Universit Miami, F L 33199 November 19, 1986

x

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 1

Development of Photoactivated Compounds as Pesticides James R. Heitz Department of Biochemistry, Mississippi Agricultural and Forestry Experiment Station, Mississippi State University, Mississippi State, MS 39762

Although ligh toxic reaction exploited until after 1970 to any great extent. The greatest concentration of effort has been in the study of photodynamically active dyes, p r i marily the halogenated fluorescein series, as prospective insecticides. More recently, compounds of plant origin have been isolated, identified, and studied as phototoxins against a wide range of pests, including insects, fungi, and weeds. The main classes studied to this time are the furanocoumarins, thiophenes, acetylenes, extended quinones, and the chlorophyll a intermediates popularized as "laser herbicides." It is apparent that this area of research will expand in the coming years rather than retrench.

The e x p e n d i t u r e o f energy f r e q u e n t l y h e l p s t o enhance the proba b i l i t y o f s u c c e s s f u l l y r e a c h i n g one's g o a l s i n t h i s u n i v e r s e . F o r as long as c h e m i s t r y has e x i s t e d as a s c i e n c e , we have i n p u t energy, most f r e q u e n t l y heat energy, i n t o c h e m i c a l r e a c t i o n s t o make the m o l e c u l e s o r t o produce the e f f e c t s which we wanted. The use o f l i g h t energy has remained q u a n t i t a t i v e l y a minor component as a means o f energy i n p u t . T h i s has a l s o been the case w i t h the development o f the p e s t i c i d e i n d u s t r y . L i g h t energy has not been used to d r i v e t o x i c o l o g i c a l r e a c t i o n s o r t o p r o v i d e s p e c i f i c i t y f o r those r e a c t i o n s t o any g r e a t e x t e n t u n t i l the decade o f the 70's. S e v e r a l r e v i e w c h a p t e r s have been w r i t t e n c o v e r i n g i n d i v i d u a l a s p e c t s o f p h o t o d y n a m i c a l l y a c t i v e p e s t i c i d e s ( 1 - 8 ) . The purpose of t h i s c h a p t e r i s t o p r o v i d e a c h r o n o l o g i c a l treatment o f the development o f l i g h t as an i n t e g r a l p a r t o f the t o x i c o l o g i c a l a c t i o n o f s e v e r a l c l a s s e s o f p e s t i c i d e s ; and a l s o , t o show the development o f the v a r i o u s c l a s s e s o f l i g h t a c t i v a t e d p e s t i c i d e s r e l a t i v e t o each o t h e r . 0097-6156/87/0339-0001 $06.25/0 © 1987 American Chemical Society

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

2

LIGHT-ACTIVATED PESTICIDES Early History The f i r s t documented s t u d y i n which l i g h t was understood t o cause an enhancement o f a c h e m i c a l l y induced t o x i c e f f e c t was t h a t o f M a r c a c c i (9^) i n which he r e p o r t e d t h a t a l k a l o i d s were more e f f e c t i v e a g a i n s t seeds, p l a n t s , f e r m e n t a t i o n s , and amphibian eggs i n s u n l i g h t than i n the dark. Rabb (10) s u b s e q u e n t l y r e p o r t e d t h a t s u n l i g h t caused an i n c r e a s e of s e v e r a l o r d e r s o f magnitude i n the a c r i d i n e s e n s i t i z e d m o r t a l i t y o f paramecia. Paramecia exposed t o a c r i d i n e i n the dark and paramecia exposed t o the sun i n c l e a r water were not n e a r l y as v u l n e r a b l e . By 1904, J o d l b a u e r and von Tappeiner (1_1) had demonstrated the requirement f o r oxygen and had c o i n e d the term "photodynamic a c t i o n . " Much l a t e r , S p i k e s and G l a d (12) would o p e r a t i o n a l l y d e f i n e photodynamic a c t i o n as the k i l l i n g or damaging o f an organism, c e l l , or v i r u s or the c h e m i c a l m o d i f i c a t i o n o f a m o l e c u l e i n the presence o f a s e n s i t i z i n g dye and molec u l a r oxygen. One proble a c t i v a t i o n of molecule or even the death o f a l i v i n g specimen was t h a t l i g h t was not con s i d e r e d as an e x p e r i m e n t a l parameter. Therefore, i t i s d i f f i c u l t to scan the e a r l y l i t e r a t u r e f o r examples s i m p l y because the l i g h t i n t e n s i t y was u s u a l l y u n c o n t r o l l e d and u n r e p o r t e d (13-27). The f i r s t r e p o r t e d use o f photodynamic a c t i o n a g a i n s t an i n s e c t t a r g e t was t h a t o f B a r b i e r i (28) i n which Anopheles and C u l e x mosquito l a r v a e were shown to be s u s c e p t i b l e t o s o l u t i o n s o f s e v e r a l c l a s s e s o f dyes i n d i r e c t s u n l i g h t . The most a c t i v e dyes were the h a l o g e n a t e d f l u o r e s c e i n d e r i v a t i v e s , e r y t h r o s i n and rose b e n g a l , alone and i n m i x t u r e ( I ) . There were no deaths r e p o r t e d from e i t h e r d y e - t r e a t e d , n o n - l i g h t - e x p o s e d p o p u l a t i o n s or non-dyetreated, light-exposed populations. The approach l a y dormant u n t i l 1950, when Schildmacher (29) t r e a t e d Anopheles and Aedes mosquito l a r v a e w i t h a s e r i e s o f dye s o l u t i o n s and exposed them t o s u n l i g h t . C o n d u c t i n g f i e l d t e s t s i n s m a l l ponds and a t l e a s t on bomb c r a t e r l e f t over from World War I I as w e l l as i n l a b o r a t o r y t e s t s , he r e p o r t e d t h a t rose b e n g a l was more t o x i c than e r y t h r o s i n and t h a t e o s i n and f l u o r e s c e i n were i n e f f e c t i v e . S c h i l d m a c h e r a l s o made the f i r s t attempt a t the d e f i n i t i o n o f the t o x i c o l o g i c a l t a r g e t when he r e p o r t e d t h a t the midgut e p i t h e l i a l c e l l s showed c o n s i d e r a b l e damage a f t e r l i g h t exposure. F i n a l l y , he observed t h a t photodynamic a c t i o n had no e f f e c t on the mosquito f i s h (Gambusia sp.) t h a t were p r e s e n t . I n o r d e r t o put these f i n d i n g s i n t o p e r s p e c t i v e , one s h o u l d be aware o f the s t a t e o f the a r t i n p e s t i c i d e t e c h n o l o g y a t t h i s t i m e . Ware (30) l i s t e d the f o l l o w i n g as some o f the i m p o r t a n t m i l e s t o n e s d u r i n g t h i s p e r i o d . Pyrethrum was i n t r o d u c e d i n t o Kenya (1928). M e t h y l bromide (1932), p e n t a c h l o r o p h e n o l (1936), TEPP (1938), B a c i l l u s t h u r i n g i e n s i s (1938), DDT ( 1 9 3 9 ) , h e x a c h l o r o c y c l o h e x a n e (1941), 2,4-D (1942), w a r f a r i n (1944), c h l o r d a n e ( 1 9 4 5 ) , toxaphene ( 1 9 4 7 ) , m a l a t h i o n (1950), and Maneb (1950) were e i t h e r d i s c o v e r e d or i n t r o d u c e d . At t h i s t i m e , the p r i m a r y c r i t e r i o n f o r a p e s t i c i d e was i t s t o x i c i t y , as R a c h e l Carson would not w r i t e " S i l e n t S p r i n g " f o r another decade.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

1.

HEITZ

Photoactivated Compounds as Pesticides

3

D u r i n g t h i s same time a l s o , b i o c h e m i s t s and p h o t o b i o l o g i s t s became i n t e r e s t e d i n the mechanism of dye s e n s i t i z e d p h o t o o x i d a t i o n and i t s e f f e c t on l i v i n g c e l l s and c e l l u l a r components. Many e x c e l l e n t reviews have been w r i t t e n on the s u b j e c t (31-37). P h o t o s e n s i t i z a t i o n has been shown to occur by one of two mechanisms: Type I and Type I I . The i n i t i a l s t e p i n the p h o t o s e n s i t i z a t i o n process i s the a b s o r p t i o n of v i s i b l e o r UV l i g h t by the s e n s i t i z e r . I n the Type I mechanism, the e x c i t e d s e n s i t i z e r conv e r t s the s u b s t r a t e t o product v i a f r e e r a d i c a l i n t e r m e d i a t e s i n c l u d i n g oxygen. In the Type I I mechanism, the e x c i t e d s e n s i t i z e r r e a c t s by c a u s i n g the f o r m a t i o n of s i n g l e t oxygen which then r e a c t s w i t h the s u b s t r a t e , t h e r e b y c o n v e r t i n g i t t o the o x i d i z e d p r o d u c t . Dye I n s e c t i c i d e s The concept l a y dormant a g a i n u n t i l 1971, when a group at West V i r g i n i a U n i v e r s i t y , Yoho f i r s t of s e v e r a l i n v e s t i g a t i o n a c t i o n a g a i n s t the a d u l t house f l y u s i n g p r i m a r i l y the halogenated f l u o r e s c e i n s e r i e s of dyes ( 3 8 ) . These p a p e r s , based s u b s t a n t i a l l y on the d i s s e r t a t i o n o f Yoho ( 1 9 ) , compared t o x i c o l o g i c a l d a t a w i t h the parameters o f l i g h t source and i n t e n s i t y , dye s t r u c t u r e and c o n v e n t r a t i o n i n the d i e t , source o f l i g h t , and l e n g t h of l i g h t exposure (38,40). L a t e r , Yoho et_ a l . (41) s t u d i e d a s e r i e s of 14 Food, Drug and Cosmetic dyes f o r e f f i c a c y i n photodynamic t o x i c i t y to house f l y a d u l t s . I t was a l s o r e p o r t e d i n the d i s s e r t a t i o n t h a t the midgut e p i t h e l i a l c e l l s appeared t o be damaged and t h a t the e x t e r n a l symptoms a s s o c i a t e d w i t h t o x i c i t y suggested an involvement w i t h the nervous system. I t can f a i r l y be s a i d t h a t the g r e a t m a j o r i t y of the work on p h o t o s e n s i t i z i n g dyes as i n s e c t i c i d e s can be t r a c e d back to the f i r s t paper i n t h i s s e r i e s as the watershed. A f t e r i t s p u b l i c a t i o n , t h e r e came a deluge o f i n t e r e s t i n t h i s area. Graham e^t a l . (42) r e p o r t e d t h a t w i t h the methylene b l u e sens i t i z e d p h o t o t o x i c i t y o f y e l l o w mealworms, the i n t e n s i t y of s u n l i g h t was much more than r e q u i r e d t o o b t a i n adequate e f f e c t i v e n e s s . Yoho e t a l . (40) a t t r i b u t e d the lower t o x i c i t y o f methylene blue ( I I ) i n f l u o r e s c e n t l i g h t r e l a t i v e to s u n l i g h t to the poor o v e r l a p w i t h the a b s o r p t i o n spectrum i n the former case. Broome ej: a_l. (43,44) r e p o r t e d on the t o x i c i t y o f a s e r i e s o f xanthene dyes w i t h the b l a c k imported f i r e ant where m o r t a l i t y was compared w i t h dye s t r u c t u r e , i n c u b a t i o n p e r i o d i n c o n t a c t w i t h the dye, dye c o n c e n t r a t i o n i n the feed and i n the i n s e c t , c o n t i n u i t y o f l i g h t exposure, l i g h t i n t e n s i t y , and exposure time. A l t h o u g h t h e r e was no m o r t a l i t y observed i n the imported f i r e ant a f t e r 3 days o f exposure to rose b e n g a l i n the d a r k , they d i d observe an onset o f m o r t a l i t y t h a t e v e n t u a l l y r e s u l t e d i n an L T 5 0 v a l u e of 8.4 days. T h i s may be compared w i t h an L T 5 0 v a l u e of 0.7 h r f o r a d u l t f i r e ants exposed to 3800yUW/cm2 from a C o o l White f l u o r e s c e n t l i g h t a f t e r 24 h r exposure to the r o s e bengal i n the dark (Broome et^ a l . (44). T h i s o b s e r v a t i o n l e d t o the acceptance o f the dark r e a c t i o n as a second, though a d m i t t e d l y much l e s s e f f i c i e n t , t o x i c mechanism caused by c e r t a i n photodynamic dyes i n i n s e c t s . Q u a n t i t a t i v e study of the dark r e a c t i o n w i t h a d u l t l i f e stages of the b o l l w e e v i l

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

LIGHT-ACTIVATED PESTICIDES

A

B

H Br I

H H H

DYE Fluorescein Eosin Erythrosin B

Structure I

Methylene Blue

Structure I I

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

1.

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Photoactivated Compounds as Pesticides

( 4 5 ) , the face f l y ( 4 6 ) , and the house f l y (47) showed the w i d e s p r e a d o c c u r r a n c e of t h i s t o x i c mechanism. In f a c t , D a v i d and H e i t z (48) r e p o r t e d on an imported f i r e ant f i e l d c o n t r o l scheme based on a p h l o x i n B-impregnated b a i t where the c o n t r o l r e p o r t e d was almost c e r t a i n l y due to the dark mechanism w o r k i n g deep w i t h i n the n e s t . At about t h i s same time, mechanism s t u d i e s were a p p e a r i n g . The a c e t y l c h o l i n e s t e r a s e from the b l a c k imported f i r e ant (49) and the b o l l w e e v i l (J>0) was s u s c e p t i b l e t o d y e - s e n s i t i z e d p h o t o o x i d a t i o n i n v i t r o but l e v e l s were not depressed i n i n s e c t s k i l l e d by photodynamic a c t i o n . Weaver e£ a l . (_51) r e p o r t e d t h a t i n the c o c k r o a c h , photodynamic a c t i o n caused a s i g n i f i c a n t decrease i n the hemolymph volume and a l a r g e i n c r e a s e i n the c r o p volume. L a t e r , Weaver ej: a l . (52) showed t h a t e r y t h r o s i n B - s e n s i t i z e d photodynamic a c t i o n caused a r e d u c t i o n of hemocytes r e l a t i v e to c o n t r o l s . At the h i g h e s t i n j e c t e d l e v e l s i n the d a r k , t h e r e was a l s o observed a r e d u c t i o n i n hemocytes mechanism. I n the absenc t h a t i n b o l l w e e v i l s fed r o s e bengal d u r i n g l a r v a l development, t h e r e were decreases i n the wet w e i g h t , dry w e i g h t , p r o t e i n l e v e l s , and l i p i d l e v e l s of the a d u l t i n s e c t . L a t e r , Callaham et^ a l . (54) showed t h a t the lower l e v e l s were due t o a l a c k of growth a f t e r a d u l t emergence i n the t r e a t e d i n s e c t s . T h i s was i n t e r p r e t e d as an energy d r a i n caused by the presence of the dye i n the a d u l t t i s s u e . In 1978, Fondren et^ al^. (47) compared the t o x i c i t i e s o f 6 xanthene dyes to the house f l y i n terms of both d i e t a r y and t i s s u e l e v e l s of the dyes i n q u e s t i o n . I n d i c a t i o n s of f e e d i n g i n h i b i t i o n were observed a t h i g h dye c o n c e n t r a t i o n s i n the food. Although s p e c i e s d i f f e r e n c e s were observed when the house f l y d a t a was compared w i t h s i m i l a r b o l l w e e v i l d a t a , i t was r e p o r t e d t h a t , i n g e n e r a l , the e f f e c t i v e n e s s of the dyes was most dependent on the phosphorescence quantum y i e l d than any o t h e r p h y s i c o - c h e m i c a l parameter. S i m i l a r i n t e r p r e t a t i o n s were made i n a l a t e r s t u d y o f the face f l y ( 4 6 ) . I n a s t u d y of l i g h t i n t e n s i t y as a c r i t i c a l parameter i n the photodynamic t o x i c i t y of rose b e n g a l to the a d u l t house f l y , Fondren and H e i t z (55) showed t h a t the accumulated number of photons needed to k i l l 50% o f a p o p u l a t i o n decreased as the i n t e n s i t y increased. T h i s would i n f e r t h a t t h e r e i s a r e g e n e r a t i v e c a p a c i t y w i t h i n the i n s e c t t h a t i s more e f f i c i e n t l y overcome by photodynamic a c t i o n as the l i g h t i n t e n s i t y i n c r e a s e s . L i g h t source was a l s o s t u d i e d as an e x p e r i m e n t a l parameter (56) where i t was shown t h a t s u n l i g h t was more e f f i c i e n t than f l u o r e s c e n t l i g h t due to the l a r g e r number of photons s t r i k i n g the t a r g e t ; but i t was a l s o shown t h a t f l u o r e s c e n t l i g h t was more e f f i c i e n t than s u n l i g h t due to the b e t t e r o v e r l a p o f the lamp output w i t h the a b s o r p t i o n spectrum of the xanthene dyes. L a v i a l l e and Dumortier (57) r e p o r t e d t h a t methylene b l u e - f e d l a r v a e of the cabbage b u t t e r f l y were s u s c e p t i b l e t o photodynamic a c t i o n a f t e r exposure to e i t h e r f l u o r e s c e n t l i g h t or s u n l i g h t . M o r t a l i t y was shown to be dependent on dye c o n c e n t r a t i o n , l i g h t i n t e n s i t y , d u r a t i o n , and w a v e l e n g t h . I n l a b o r a t o r y t o x i c i t y t e s t s u s i n g s e v e r a l xanthene dyes a g a i n s t the b l a c k cutworm, Clement et a l . (58) found t h a t rose b e n g a l was

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the most e f f e c t i v e and t h a t t o x i c i t y was d i r e c t l y dependent on the light intensityI n t h i s c a s e , the l a r v a e a v o i d s the l i g h t and t h a t makes t h i s p a r t i c u l a r a p p l i c a t i o n u n d e s i r e a b l e . C r e i g h t o n et a l . (59) r e p o r t e d on the t o x i c i t y of rose bengal t o the cabbage l o o p e r , the c o r n earworm, and the p i c k l e w o r m . Photodynamic a c t i o n was r e l a t i v e l y i n e f f e c t i v e under these c o n d i t i o n s , but the dark t o x i c i t y was observed. I n 1979, P i m p r i k a r ^ t a l . (60) began r e p o r t i n g on an extended s e r i e s o f t e s t s w i t h mosquito l a r v a e . Under f l u o r e s c e n t l i g h t and at r o s e bengal treatment l e v e l s of 1 t o 20 ppm, C u l e x l a r v a e were more s u s c e p t i b l e than Aedes l a r v a e and e a r l y i n s t a r s were more s u s c e p t i b l e than l a t e r i n s t a r s . P h y s i o l o g i c a l and m o r p h o l o g i c a l a b n o r m a l i t i e s were observed i n the pupal and a d u l t stage a f t e r l a r v a l stage treatment which suggested improper c h i t i n f o r m a t i o n i n the i n s e c t . T h i s sometimes r e s u l t e d i n i n c o m p l e t e e x t r i c a t i o n o f the pupal stage from the l a r v a l c u t i c l e and of the a d u l t stage from the pupal c u t i c l e . Wher They a l s o r e p o r t e d the observanc s i m i l a r t o those observed a f t e r treatment w i t h i n s e c t growth r e g u lators • P i m p r i k a r et^ al. (61) attempted t o c o n t r o l house f l i e s i n a commercial caged l a y e r house u s i n g weekly a p p l i c a t i o n s of aqueous s o l u t i o n s of e r y t h r o s i n B d i r e c t l y on the manure. I n a d u p l i c a t e d 5 week treatment p e r i o d , they r e p o r t e d decreases o f a d u l t and l a r v a l house f l i e s up t o 90% w i t h r e s p e c t t o p r e t r e a t m e n t l e v e l s w i t h no change i n the b e n e f i c i a l s o l d i e r f l y l a r v a l p o p u l a t i o n . The dye was r e p o r t e d t o be somewhat r a p i d l y degraded i n the manure i l l u minated by i n d i r e c t s u n l i g h t such t h a t o n l y about 20% was e x t r a c t a b l e 1 week a f t e r s p r a y i n g . As a r e s u l t o f these t e s t s , P i m p r i k a r et a l . (62) s t u d i e d the e f f e c t s of s e v e r a l f l u o r e s c e i n d e r i v a t i v e s on each developmental stage o f the house f l y . Treated adults e x h i b i t e d lowered f e c u n d i t y , the eggs e x h i b i t e d a reduced v i a b i l i t y , and m o r t a l i t y was observed i n each l i f e stage o f the house f l y . C a r p e n t e r and H e i t z (63) showed t h a t when l a r v a l mosquitoes were exposed t o r o s e bengal and v i s i b l e l i g h t , s i g n i f i c a n t acute m o r t a l i t y was observed. F u r t h e r , i f the t r e a t e d mosquitoes were i l l u m i n a t e d w i t h v i s i b l e l i g h t and then put i n t o d a r k n e s s , a l a t e n t m o r t a l i t y was observed. The l i g h t treatment was n e c e s s a r y t o o b t a i n the l a t e n t m o r t a l i t y , as the c o n t r o l s exposed t o the same dye c o n c e n t r a t i o n s i n the dark e x h i b i t e d no l a t e n t m o r t a l i t y . When the l a t e n t m o r t a l i t y was added t o the acute m o r t a l i t y , i t was observed t h a t the t o t a l t o x i c i t y o f the rose bengal was i n c r e a s e d by 1 0 - f o l d over the dark t o x i c i t y . L a t e r , C a r p e n t e r and H e i t z (64) s t u d i e d the r e l a t i o n s h i p s between the slow, l i g h t - i n d e p e n d e n t mechanism, the r a p i d , l i g h t - d e p e n d e n t mechanism, and the s l o w , l i g h t - i n i t i a t e d , l a t e n t mechanism d u r i n g the treatment of C u l e x l a r v a e w i t h e r y t h r o s i n B. Q u a n t i t a t i v e a n a l y s i s was hampered by the p h o t o d e g r a d a t i o n o f the e r y t h r o s i n B d u r i n g the time course o f the study which made the e x p r e s s i o n of t o x i c i t y r e l a t i v e t o dye concentration impossible.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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F a i r b r o t h e r et a l . (j>5) made a v e r y complete study o f the t o x i c o l o g i c a l e f f e c t s o f e r y t h r o s i n B and rose bengal on the face f l y When l a r v a e developed on manure i n t o which e i t h e r dye was i n c o r p o r a t e d , e i t h e r by hand or by passage o f the dye through c a t t l e , m o r t a l i t y was observed at each l i f e s t a g e . Some of the f l i e s d i e d at v a r i o u s stages o f emergence as i f the e f f o r t a s s o c i a t e d w i t h emergence was too s t r e s s f u l . S e v e r a l of the a d u l t s were unable to complete the e x t r i c a t i o n from the puparium and were s t u c k t o the c h i t i n i n n e r l i n i n g of the puparium. Others had deformed wings. A d u l t s , h e l d from emergence and i l l u m i n a t e d w i t h v i s i b l e l i g h t , were observed to have a much h i g h e r m o r t a l i t y than c o n t r o l s , thus s u g g e s t i n g t h a t dye s e q u e s t e r e d i n the i n s e c t body d u r i n g d e v e l o p ment from l a r v a e to a d u l t was r e s p o n s i b l e f o r the t o x i c i t y . This i s the f i r s t r e p o r t of photodynamic a c t i o n o c c u r r i n g i n a l i f e stage d i f f e r e n t from the l i f e stage which i n g e s t e d the dye. C a r p e n t e r ejt al. (66) r e p o r t e d t h a t the presence of f l u o r e s c e i n enhanced the t o x i c i t y o Aedes l a r v a e . T h i s s y n e r g i s z a t i o n of photons absorbed by the f l u o r e s c e i n m o l e c u l e t h a t were not of the proper wavelength f o r a b s o r p t i o n by the r o s e b e n g a l m o l e c u l e . A U n i t e d S t a t e s p a t e n t was i s s u e d c o v e r i n g the s y n e r g i s m of a n o n t o x i c dye w i t h a demonstrated t o x i c dye i n b o t h house f l y and mosquito systems (6i7). L a t e r , i n t e s t s i n v o l v i n g 8 xanthene dyes, i t was not p o s s b i l e to c o n f i r m the mechanism of a c t i o n as t h a t r e f e r r e d t o above ( 6 8 ) . F u r t h e r , the s y n e r g i s m c o u l d not be c o r r e l a t e d w i t h the number of h a l o g e n s , p e r c e n t h a l o g e n a t i o n , molec u l a r w e i g h t , p a r t i t i o n c o e f f i c i e n t , f l u o r e s c e n c e quantum y i e l d o f the s y n e r g i s t dye, or the o v e r l a p i n t e r v a l f o r the s y n e r g i s t dye w i t h e r y t h r o s i n B. The mechanism o f a c t i o n of the s y n e r g i s m observed w i t h the xanthene dyes i s s t i l l u n e x p l a i n e d . S a k u r a i and H e i t z (69) s t u d i e d the i n h i b i t i o n of growth and the photodynamic a c t i o n caused by r o s e b e n g a l and e r y t h r o s i n B i n the house f l y . L a r v a e r e a r e d i n the dark on agar c o n t a i n i n g e i t h e r dye e x h i b i t e d a c o n c e n t r a t i o n dependent decrease i n p u p a t i o n r a t e and i n a d u l t emergence. House f l i e s which had consumed a n o n l e t h a l amount of dye i n the l a r v a l stage e x h i b i t e d a c o n s i d e r a b l e l i g h t dependent t o x i c i t y i n the a d u l t s t a g e . I t was a l s o observed t h a t pupae i n j e c t e d w i t h rose b e n g a l developed i n t o a d u l t s which were more s u s c e p t i b l e to photodynamic a c t i o n than a d u l t s i n j e c t e d w i t h the same dye. F u r t h e r , the s u s c e p t i b i l i t y of the i n j e c t e d a d u l t s was comparable to a d u l t s fed the dye, thus s u g g e s t i n g t h a t the a l i mentary c a n a l may not be the o n l y s i t e o f a c t i o n as suggested p r e v i o u s l y (29,39,51). I n 1983, R e s p i c i o and H e i t z (70) began a study of the d e v e l o p ment of r e s i s t a n c e to e r y t h r o s i n B i n the house f l y . A l a b o r a t o r y s t r a i n developed o n l y 6 - f o l d r e s i s t a n c e a f t e r 40 g e n e r a t i o n s of c h a l l e n g e by e r y t h r o s i n B. T h i s low l e v e l o f r e s i s t a n c e was due to the i n b r e d q u a l i t y o f the l a b o r a t o r y s t r a i n . A new, w i l d s t r a i n developed a 4 8 - f o l d r e s i s t a n c e a f t e r 32 g e n e r a t i o n s of exposure to i n c r e a s i n g l e v e l s of e r y t h r o s i n B i n the d i e t . Upon removal of the s e l e c t i o n p r e s s u r e f o r 20 g e n e r a t i o n s , the r e s i s t a n c e remained constant. R e c i p r o c a l c r o s s e s showed t h a t the r e s i s t a n c e i s i n h e r i t e d as a codominant c h a r a c t e r and t h a t sex l i n k a g e i s not

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i n v o l v e d . L a t e r , the c r o s s - r e s i s t a n c e of e r y t h r o s i n B - r e s i s t a n t house f l i e s was s t u d i e d a g a i n s t s t r a i n s r e s i s t a n t to propoxur, DDT, p e r m e t h r i n , and d i c h l o r v o s (71). No c r o s s - r e s i s t a n c e f o r a d i f f e r e n t p e s t i c i d e was observed f o r any of the 5 s t r a i n s , w i t h one exception. The e r y t h r o s i n B - r e s i s t a n t s t r a i n was c r o s s - r e s i s t a n t to p h l o x i n B and r o s e b e n g a l , but t h i s i s t o be expected s i n c e they f u n c t i o n by the same mechanism. R e c e n t l y , c r o s s - r e s i s t a n c e has been shown when the e r y t h r o s i n B - r e s i s t a n t s t r a i n was c h a l l e n g e d by a l p h a - t e r t h i e n y l mediated photodynamic a c t i o n ( P i m p r i k a r , G.D. and H e i t z , J.R., u n p u b l i s h e d r e s u l t s ) . The r e l a t i v e t o x i c i t i e s of 6 xanthene dyes t o C u l e x and Aedes mosquito l a r v a e was r e p o r t e d by P i m p r i k a r e t a l . ( 7 2 ) . Rose b e n g a l was the most t o x i c f o l l o w e d by p h l o x i n B and e r y t h r o s i n B. At the same t i m e , i t was shown t h a t these same dyes e x h i b i t e d a low t o x i c i t y t o the mosquito f i s h , t h e r e b y c o n f i r m i n g the o b s e r v a t i o n o f Schildmacher ( 2 9 ) , and d i d not a f f e c t the p r e d a t o r y e f f i c i e n c y o f the f i s h . The l a c k of a would a l l o w the dyes t ment scheme. I n 1984, P i m p r i k a r and H e i t z (73) observed an u n u s u a l l y h i g h i n s e c t i c i d a l a c t i v i t y i n Aedes mosquito l a r v a e which had been i l l u minated a f t e r exposure to the i n s o l u b l e f r e e a c i d forms o f the xanthene dyes. I n a l l p r e v i o u s s t u d i e s , the l a r v a e had been t r e a t e d w i t h the water s o l u b l e s a l t forms o f the dyes and the l a r v a e consumed the dye as they i n g e s t e d the w a t e r . W i t h the i n s o l u b l e dyes, they were a b l e t o f i l t e r feed on dye p a r t i c l e s and t h e r e b y r e c e i v e a h i g h e r l e v e l of dye. T o x i c i t y r a t i o s ranged up to 2 o r d e r s of magnitude between the s o l u b l e and i n s o l u b l e forms of the same dye. In a l a t e r s t u d y , C a r p e n t e r et a l . (74) showed t h a t the i n s o l u b l e forms of the xanthene dyes were 1 0 - f o l d more e f f e c t i v e a g a i n s t C u l e x mosquito l a r v a e than the s o l u b l e forms. F u r t h e r , they r e p o r t e d t h a t when the i n s o l u b l e forms o f the dyes were d i s p e r s e d w i t h a s u r f a c t a n t , such as sodium l a u r y l s u l f a t e , the dyes were 50- t o 6 0 - f o l d more e f f e c t i v e than the s o l u b l e forms. R e s p i c i o et a l _ . (75^) s t u d i e d the t o x i c i t y t o C u l e x mosquito l a r v a e of c o p r e c i p i t a t e d f r e e a c i d , n o n d i s p e r s i b l e and d i s p e r s i b l e f o r m u l a t i o n s of f l u o r e s c e i n and e r y t h r o s i n B. The 1:1 c o m b i n a t i o n o f f l u o r e s c e i n : e r y t h r o s i n B, d i s p e r s e d w i t h sodium d o d e c y l s u l f a t e , was the most t o x i c f o r m u l a t i o n and a l s o showed s y n e r g i s t i c a c t i o n . I n a more d e t a i l e d study of the s y n e r g i s t i c e f f e c t , they showed t h a t the 1:1 m i x t u r e of f l u o r e s c e i n : r o s e b e n g a l was more t o x i c than the 3:1 m i x t u r e , but the 3:1 m i x t u r e e x h i b i t e d more s y n e r g i s m ( R e s p i c i o , N.C., C a r p e n t e r , T.L., and H e i t z , J.R. J . M i s s . Acad. S c i , i n p r e s s ) . C a r p e n t e r e t a l . (76) e v a l u a t e d a s e r i e s o f 8 d i s p e r s a n t s f o r use w i t h the i n s o l u b l e forms o f the dyes and none were t o x i c a l o n e . E r y t h r o s i n B, d i s p e r s e d w i t h sodium d o d e c y l s u l f a t e , was the most t o x i c a g a i n s t C u l e x mosquito l a r v a e . In s m a l l - s c a l e f i e l d t e s t s , t h i s f o r m u l a t i o n caused s i g n i f i c a n t r e d u c t i o n s i n l a r v a l and emergent a d u l t p o p u l a t i o n s o f C u l e x mosquitoes a t c o n c e n t r a t i o n s r a n g i n g from 0.25 t o 8.0 ppm. Not a l l of the work w i t h the f l u o r e s c e i n dyes concerned i n s e c t s . In 1985, Knox and Dodge (7J7) r e p o r t e d on the photodynamic

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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9

a c t i o n of e o s i n on pea l e a f t i s s u e . C h l o r o p l a s t s were shown t o be p a r t i c u l a r l y s e n s i t i v e to v i s i b l e l i g h t a f t e r e o s i n t r e a t m e n t . The t r e a t e d t i s s u e e x h i b i t e d lowered p h o t o s y n t h e t i c oxygen e v o l u t i o n , lowered p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t c a p a b i l i t y , lowered l e v e l s of r i b u l o s e - b i s p h o s p h a t e c a r b o x y l a s e and NADPH-dependent g l y c e r a l d e h y d e - 3 - p h o s p h a t e dehydrogenase, and pigment l o s s . The i n i t i a l l o s s of p h o t o s y n t h e t i c a c t i v i t y was a s s o c i a t e d w i t h damage t o the t h y l a k o i d membranes. In an accompanying paper, Knox and Dodge (78) f u r t h e r c h a r a c t e r i z e d the s i t e of the photodynamic a c t i o n i n pea l e a f t i s s u e as photosystem I I . Robins and Beatson (_79) attempted t o p r o t e c t house f l y l a r v a e against e r y t h r o s i n B s e n s i t i z e d p h o t o t o x i c i t y . Beta-carotene prot e c t e d , but b u t y l a t e d h y d r o x y t o l u e n e , a s c o r b a t e , and d i a z a b i c y c l o o c t a n e a c t u a l l y enhanced the t o x i c e f f e c t . Hawkins et_ a l . (80) showed t h a t e r y t h r o s i n B and v i s i b l e l i g h t (from e i t h e r f l u o r e s c e n t sources or s u n l i g h t ) were t o x i c to the i n f e c t i o u s 3rd stage l a r v a naturally infected cattle c o n s e c u t i v e d a i l y o r a l t r e a t m e n t s of the c a t t l e . L a t e r , they r e p o r t e d t h a t the photodynamic a c t i o n was i n e f f e c t i v e a g a i n s t the a d u l t stage v i a b i l i t y or f e c u n d i t y (Hawkins, J.A.; J o h n s o n - D e l i v o r i a s , M.H.; H e i t z , J.R. Veterin. Parasitol., in p r e s s ) . There was a c o n s i s t e n t e f f e c t on the 3rd stage l a r v a e which was dependent upon dosage, time of l i g h t exposure and, t o a l e s s e r e x t e n t , the l e n g t h o f time the l a r v a e were l e f t i n the presence of the dye. P h o t o a c t i v e P l a n t Components I n the study of the p h o t o a c t i v e dyes, the r e s e a r c h was focused p r i m a r i l y on a deep u n d e r s t a n d i n g of the mechanisms of a c t i o n of o n l y a s m a l l number of dyes from p r e d o m i n a t e l y one c l a s s o f compounds. I n the study o f p h o t o a c t i v e p l a n t components, t h e r e has been a s h i f t of emphasis. Much of the r e s e a r c h has been aimed a t i s o l a t i o n and i d e n t i f i c a t i o n of n o v e l p l a n t components, d e l i n e a t i o n of the g e n e r a l mechanisms of a c t i o n and the type of s e n s i t i v e organism. As such, t h e r e are fewer papers on any g i v e n compound, but many more compounds s t u d i e d . Some o f the major c l a s s e s o f p l a n t d e r i v e d compounds w i l l be examined h e r e . R e c e n t l y , an e n t i r e i s s u e of the J o u r n a l of Chemical E c o l o g y was devoted t o the i n v i t e d papers p r e sented a t a symposium on i n t e r a c t i o n s between i n s e c t s and photoa c t i v e p l a n t s p r e s e n t e d a t the 1984 n a t i o n a l meeting o f the E n t o m o l o g i c a l S o c i e t y of America ( 8 1 ) . S e v e r a l r e l a t e d papers were a l s o i n c l u d e d which were not p a r t o f the symposium. Furanocoumarins Furanocoumarin8 have been i m p l i c a t e d i n c e r t a i n p h o t o t o x i c r e s p o n ses i n g r a z i n g c a t t l e ( 8 2 ) . I n 1978, Berenbaum (83) r e p o r t e d t h a t when the l i n e a r furanocoumarin, x a n t h o t o x i n ( I I I ) , was a d m i n i s t e r e d to the l a r v a e of the s o u t h e r n armyworm, a low l e v e l of t o x i c i t y was observed t h a t was g r e a t l y enhanced when UV l i g h t was shown upon the l a r v a e . She a l s o observed a l o n g e r time r e q u i r e d f o r p u p a t i o n t o

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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o c c u r i n those l a r v a e t h a t d i d n o t d i e - The b i o l o g i c a l a c t i v i t y o f the furanocoumarins a r e due t o the i n t e r c a l c a t i o n o f the m o l e c u l e i n t o t h e double s t r a n d e d DNA where, upon a c t i v a t i o n by UV l i g h t , c o v a l e n t bonds a r e formed w i t h p y r i m i d i n e bases ( 8 4 ) . Song and T a p l e y (85) demonstrated t h a t the mechanism o f a c t i o n was Type I i n which oxygen r a d i c a l s a r e i n v o l v e d . L a t e r , Berenbaum and Feeny (86) r e p o r t e d t h a t the a n g u l a r f u r a n o c o u m a r i n , a n g e l i c i n ( I V ) , reduced the growth r a t e and the f e c u n d i t y of the l a r v a e o f the b l a c k s w a l l o w t a i l b u t t e r f l y , whereas x a n t h o t o x i n was n o t a p p r e c i a b l y t o x i c t o t h i s i n s e c t . I t was i n t e r p r e t e d t h a t t h e a n g u l a r forms o f the furanocoumarin were l a t e r e v o l u t i o n a r y developments which h e l p e d t o p r o t e c t t h e p l a n t from i n s e c t h e r b i v o r y . R e c e n t l y , I v i e et^ a l . (87) i n an i n i t i a l r e p o r t on the m e t a b o l i s m o f furanocoumarins by b l a c k s w a l l o w t a i l b u t t e r f l y l a r v a e , showed t h a t t h i s i n s e c t d e t o x i f i e s t h i s c l a s s o f compounds by m e t a b o l i s m i n t h e midgut t i s s u e p r i o r t o a b s o r p t i o n . I n t h i s manner, a p p r e c i a b l not e n t e r t h e body c i r c u l a t i o n i n c r e a s e d p h o t o t o x i c i t y o f t h e a n g u l a r furanocoumarins r e l a t i v e t o the l i n e a r furanocoumarins was due t o a s l o w e r r a t e o f h y d r o l y s i s of the f u r a n r i n g o f the a n g u l a r d e r i v a t i v e s (88,89). Ashwood-Smith et^ al. (90) r e p o r t e d t h a t the b l a c k s w a l l o w t a i l l a r v a e were a b l e t o degrade x a n t h o t o x i n i n t o b i o l o g i c a l l y i n a c t i v e compounds. The enzyme r e a c t i o n r e q u i r e d an e l e c t r o n g e n e r a t i n g and a c c e p t i n g system s i m i l a r t o t h e mixed f u n c t i o n o x i d a s e s o f mamm a l i a n microsomes. I r i v i t r o s t u d i e s o f t h e r e l a t i v e m e t a b o l i c r a t e s o f h y d r o l y s i s o f x a n t h o t o x i n by homogenates o f l a s t stage l a r v a e o f the b l a c k s w a l l o w t a i l b u t t e r f l y and the f a l l armyworm showed t h a t the former i n s e c t h y d r o l y z e d the x a n t h o t o x i n 6 times f a s t e r than the l a t t e r i n s e c t ( 9 1 ) . Alpha-Terthienyl

and P o l y a c e t y l e n e s

A l p h a - t e r t h i e n y l (V) was shown t o be n e m a t i c i d a l by Uhlenbroek and B i j l o o (92). Gommers (93^) r e p o r t e d t h a t i r r a d i a t i o n w i t h near UV l i g h t s t r o n g l y enhanced t h e n e m a t i c i d a l a c t i v i t y o f a l p h a t e r t h i e n y l . L a t e r , Gommers and G e e r l i g s (94) showed t h a t endoparas i t i c p l a n t nematodes which had been exposed t o a l p h a - t e r t h i e n y l i n the r o o t s o f A f r i c a n m a r i g o l d s f o r 10 days were r a p i d l y k i l l e d upon exposure t o near UV l i g h t . Bakker e t a l . (95) demonstrated t h a t , upon i r r a d i a t i o n , a l p h a - t e r t h i e n y l g e n e r a t e s a r e a c t i v e oxygen spec i e s , p r o b a b l y s i n g l e t oxygen, upon which the n e m a t i c i d a l a c t i v i t y depends. Gommers et^ a l . (96) r e p o r t e d t h a t t h e i r r a d i a t i o n o f a l p h a - t e r t h i e n y l r e q u i r e d a e r o b i c c o n d i t i o n s f o r the r a p i d k i l l i n g of nematodes. I n v i t r o s t u d i e s o f enzyme i n h i b i t i o n and p r o t e c t i o n by a s e r i e s o f s i n g l e t oxygen quenchers f u r t h e r supported t h e h y p o t h e s i s t h a t the a c t i v e oxygen formed i n t h e r e a c t i o n was s i n g l e t oxygen. A l p h a - t e r t h i e n y l and p h e n y l h e p t a t r i y n e (VI) were shown t o be powerf u l t o x i c p h o t o s e n s i t i z e r s a g a i n s t f i r s t and f o u r t h i n s t a r Aedes mosquito and b l a c k f l y l a r v a e i n b o t h s u n l i g h t and UV l i g h t ( 9 7 , 9 8 ) . The mode o f a c t i o n o f a l p h a - t e r t h i e n y l was shown t o be photodynamic i n nature but that of phenylheptatriyne-type compounds was n o t as

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

1.

HEITZ

11

Photoactivated Compounds as Pesticides

OCH

3

Xanthotoxin

Structure I I I

Angelicin

S t r u c t u r e IV

a -Terthienyl

Structure V

^Q^"C=C-C»C-C=C-CH

3

I-Phenyl-1,3,5-heptatriyne

S t r u c t u r e VI

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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c l e a r (99) . McLachlan e t a_l. (100) i n v e s t i g a t e d s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s f o r a s e r i e s of p o l y a c e t y l e n e and thiophene d e r i v a t i v e s a g a i n s t a b a c t e r i u m and a y e a s t w i t h the thiophenes b e i n g g e n e r a l l y more t o x i c than the a c e t y l e n e s . A c t i v i t y was d i r e c t l y dependent upon the number of thiophene r i n g s and a c e t y l e n e bonds. There was a p o s i t i v e c o r r e l a t i o n between p h o t o t o x i c i t y and the o c t a n o l - w a t e r p a r t i t i o n c o e f f i c i e n t ; but t h e r e was l i t t l e c o r r e l a t i o n w i t h photon a b s o r p t i o n . The dose response i n r e l a t i o n t o the l i g h t source was s t u d i e d by Arnason e t a l . ( 1 0 1 ) . A l t h o u g h a l p h a - t e r t h i e n y l e x h i b i t e d low t o x i c i t y i n the absence o f l i g h t , the enhanced t o x i c i t y t o Aedes mosquito l a r v a e upon i r r a d i a t i o n by near UV l i g h t l e d t o i t s i n v e s t i g a t i o n as a commercial l a r v i c i d e i n f i e l d t r i a l s u s i n g simul a t e d s m a l l ponds. A l p h a - t e r t h i e n y l was even more t o x i c t o the mosquito l a r v a e i n s u n l i g h t . An a c t i o n spectrum showed t h a t t h e r e was good agreement between l i g h t a b s o r p t i o n and t o x i c o l o g i c a l action. I n 1983, Kagan and Cha t a t r i y n e and a l p h a - t e r t h i e n y l d i s p l a y e d o v i c i d a l a c t i v i t y a g a i n s t the eggs o f the f r u i t f l y i n the d a r k . They r e p o r t e d t h a t i r r a d i a t i o n by l o n g wavelength UV l i g h t enhanced the t o x i c i t y by 37and 4 3 3 3 - f o l d , r e s p e c t i v e l y . U s i n g the s i n g l e t oxygen dependent c o n v e r s i o n of adamantylidene adamantane to adamantanone, Kagan et_ a l . (103) were a b l e t o compare the r e l a t i v e s i n g l e t oxygen g e n e r a t i n g c a p a b i l i t y o f a s e r i e s o f thiophene d e r i v a t i v e s . The p o l y a c e t y l e n i c compound, c i s - d e h y d r o m a t r i c a r i a e s t e r was shown t o be o v i c i d a l t o f r e s h l y l a i d eggs of the f r u i t f l y . Upon i r r a d i a t i o n w i t h u l t r a v i o l e t l i g h t the o v i c i d a l a c t i v i t y was enhanced (104). L a t e r , Downum ej: a l . (105) r e p o r t e d t h a t the tobacco hornworm, when g i v e n a s i n g l e i n g e s t e d dose o f a l p h a - t e r t h i e n y l f o l l o w e d by exposure t o UV l i g h t , e x h i b i t e d delayed and abnormal pupal f o r m a t i o n w i t h no subsequent a d u l t emergence. T o p i c a l a p p l i c a t i o n o f a l p h a - t e r t h i e n y l f o l l o w e d by i r r a d i a t i o n w i t h near UV l i g h t a f f e c t e d b o t h the s c l e r o t i z a t i o n and m e l a n i z a t i o n of the pupal case i n l a t e r development. Kagan et^ a l . (106) demonstrated the f i r s t example of the i n a c t i v a t i o n o f a c e t y l c h o l i n e s t e r a s e in v i v o by a p h o t o a c t i v e p e s t i c i d e when they showed t h a t a l p h a - t e r t h i e n y l , as w e l l as 3 i s o m e r s , caused the i n h i b i t i o n o f t h i s enzyme i n Aedes mosquito l a r v a e upon UV l i g h t i r r a d i a t i o n . L a t e r , Reyftmann e_t a l . (107) showed t h a t a l p h a - t e r t h i e n y l e x h i b i t e d a very l o n g - l i v e d e x c i t e d t r i p l e t s t a t e which a l l o w e d i t t o r e a c t v e r y f a v o r a b l y w i t h oxygen, thereby p r o d u c i n g s i n g l e t oxygen. S i n c e i t does not r e a c t w e l l w i t h hydrogen or e l e c t r o n donors, i t appears t h a t a l p h a - t e r t h i e n y l f u n c t i o n s p r i m a r i l y as a Type I I photodynamic agent. Kagan ej: a l . (108) s t u d i e d the p h o t o t o x i c e f f e c t s o f a l p h a t e r t h i e n y l on f a t h e a d minnows and i t was found t o be at l e a s t t w i c e as potent as rotenone and n e a r l y as potent as e n d r i n . I n 1985, a Canadian p a t e n t was awarded to Towers et_ a l . (109) c o v e r i n g the c o n t r o l of p e s t s ( a l g a e , f u n g i , nematodes, or h e r b i v o r o u s i n v e r t e b r a t e s ) by p o l y a c e t y l e n e s • A c t i v a t i o n of the p o l y a c e t y l e n e by the UV component of s u n l i g h t enhanced the t o x i c e f f e c t s observed i n the absence o f l i g h t .

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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H y p e r i c i n and

13

Cercosporin

I t has been known f o r many y e a r s , t h a t when g r a z i n g animals feed on c e r t a i n members of the p l a n t genus Hypericum, they become s e n s i t i v e t o s u n l i g h t . T h i s s e n s i t i v i t y i s accompanied by i n t e n s e s k i n i r r i t a t i o n and i n f l a m m a t i o n which may become f a t a l . H o r s l e y (110) showed t h a t t h i s c o n d i t i o n was caused by h y p e r i c i n ( V I I ) , a h i g h l y condensed quinone (111,112). Yamazaki e_t a_l. (113) noted the s i m i l a r i t i e s between the s t r u c t u r e s of h y p e r i c i n ( V I I ) and c e r c o s p o r i n ( V I I I ) . When they exposed c e r c o s p o r i n - t r e a t e d mice and b a c t e r i a t o l i g h t , m o r t a l i t y was observed. C e r c o s p o r i n a l s o was shown t o damage p l a n t t i s s u e under i l l u m i n a t i o n by i n c a n d e s c e n t l i g h t ( 1 1 4 ) . Daub (115) r e p o r t e d t h a t the k i n e t i c s of the k i l l i n g of tobacco p l a n t c e l l s was a f u n c t i o n o f c e r c o s p o r i n c o n c e n t r a t i o n , l i g h t i n t e n s i t y , l i g h t w a v e l e n g t h , and s i n g l e t oxygen quenchers. S i n c e the t o x i c response was i n h i b i t e d by Dabco and b i x i n , known quenchers of s i n g l e t oxygen produced s i n g l e t oxyge agent. L a t e r , Daub (116) showed t h a t c e r c o s p o r i n - c a u s e d e l e c t r o l y t e leakage from tobacco l e a f d i s c s was p r o b a b l y due t o l i p i d h y d r o p e r o x i d e f o r m a t i o n from membrane l i p i d s . Cercosporin was shown to o x i d i z e s o l u t i o n s of methyl l i n o l e n a t e , w h i l e a l p h a t o c o p h e r o l had an i n h i b i t o r y e f f e c t on the c e r c o s p o r i n - m e d i a t e d l i p i d p e r o x i d a t i o n . Daub and B r i g g s (117) then showed t h a t the u n s a t u r a t e d a c y l c h a i n s o f l i p i d s were the t a r g e t o f the photodynamic a c t i o n . When the u n s a t u r a t e d a c y l c h a i n s are o x i d i z e d , s p i n l a b e l l i n g experiments showed t h a t the membranes become more r i g i d a t a l l temperatures and t h a t the membrane phase t r a n s f o r m a t i o n temperature i n c r e a s e d from 12.7° t o 20.8°C. I n 1983, Daub and H a n g a r t e r ( 1 1 8 ) , r e p o r t e d t h a t c e r c o s p o r i n produced s u p e r o x i d e r a d i c a l s as w e l l as s i n g l e t oxygen upon exposure t o l i g h t i n the presence o f oxygen. C e r c o s p o r i n r e a c t e d w i t h c h o l e s t e r o l t o form the 5 a l p h a - h y d r o p e r o x i d e of c h o l e s t e r o l . T h i s r e a c t i o n i s s p e c i f i c f o r s i n g l e t oxygen. C e r c o s p o r i n a l s o reduced p - n i t r o b l u e t e t r a z o l i u m c h l o r i d e which i s r e a d i l y reduced by s u p e r o x i d e . Superoxide dismutase, an enzyme which r e a c t s v e r y r a p i d l y w i t h superoxide, i n h i b i t e d t h i s r e a c t i o n . In 1985, Knox and Dodge (119) i s o l a t e d h y p e r i c i n from the h a i r y St. John's wort and showed t h a t i t s e n s i t i z e d the p h o t o o x i d a t i o n of m e t h y l l i n o l e n a t e . The r e a c t i o n was i n h i b i t e d by the c a r o t e n o i d , c r o c i n . H y p e r i c i n was shown t o produce s i n g l e t oxygen due t o oxygen consumption d u r i n g the s e n s i t i z e d p h o t o o x i d a t i o n of i m i d a z o l e and a l s o due t o i n h i b i t e d r a t e s o f oxygen consumption d u r i n g the r e a c t i o n i n the presence o f deuterium o x i d e or sodium a z i d e . H y p e r i c i n a l s o caused pigment l o s s and ethane p r o d u c t i o n from pea l e a f d i s c s under l i g h t exposure. Laser Herbicides S i n c e many p e s t i c i d e s are d i s c o v e r e d as a r e s u l t of e x t e n s i v e s c r e e n i n g programs of many c a n d i d a t e c h e m i c a l s , i t i s r e a l l y not n e c e s s a r y t o understand the mechanism o f a c t i o n o f the c a n d i d a t e

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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p e s t i c i d e at f i r s t . R a t h e r , i t i s n e c e s s a r y o n l y t h a t i t be e f f e c t i v e . There does e x i s t i n the realm of the f a m i l y of p h o t o a c t i v a t e d p e s t i c i d e s , a h e r b i c i d e which was a c t u a l l y d e s i g n e d on the b a s i s of knowledge of the i n h e r e n t b i o c h e m i c a l pathways i n p l a n t s . I n 1969, E l l s w o r t h and A r o n o f f (120) i n i t i a l l y proposed t h a t c h l o r o p h y l l was b i o s y n t h e s i z e d v i a 2 a l t e r n a t e p a r a l l e l pathways i n v o l v i n g monovinyl d e r i v a t i v e s and d i v i n y l d e r i v a t i v e s . Over the next s e v e r a l y e a r s , R e b e i z and h i s coworkers s t u d i e d c h l o r o p l a s t b i o g e n e s i s i n p l a n t s (4,121). They l a t e r proposed t h a t each a l t e r nate p a r a l l e l pathway c o n t a i n e d p a r a l l e l subpathways u t i l i z i n g f u l l y e s t e r i f i e d d e r i v a t i v e s and a c i d i c d e r i v a t i v e s . They r e a l i z e d at the time t h a t the known mode of a c t i o n of no h e r b i c i d e took advantage of t h i s a s p e c t of p l a n t b i o s y n t h e s i s . If chlorophyll b i o s y n t h e s i s was used as the t a r g e t f o r the h e r b i c i d a l a c t i o n , i t would a l l o w f o r a c e r t a i n s p e c i f i c i t y . F u r t h e r , the d i v e r s i t y of c h l o r o p h y l l a b i o s y n t h e t i c pathways a l l o w e d f o r d i v e r s i t y i n design. The mechanism o the photodynamic a c t i v i t ves which are p a r t of the c h l o r o p h y l l a b i o s y n t h e t i c scheme. T h e r e f o r e , i t would be dependent on the b i o s y n t h e s i s and accumulat i o n of the t e t r a p y r r o l e s by the sprayed p l a n t t a r g e t s . F u r t h e r , a p o s t - s p r a y p e r i o d of darkness of s e v e r a l hours would be r e q u i r e d f o r the a c c u m u l a t i o n of the t e t r a p y r r o l e s . F i n a l l y , upon exposure to l i g h t , a v e r y damaging photodynamic e f f e c t , c a t a l y z e d by the a c c u mulated t e t r a p y r r o l e s , would o c c u r which w i l l r e s u l t i n the death of the p l a n t t a r g e t . I n o r d e r to s t i m u l a t e the b i o s y n t h e s i s of t e t r a p y r r o l e s i n the p l a n t t a r g e t , d e l t a - a m i n o l e v u l i n i c a c i d and 2 , 2 - d i p y r i d y l were sprayed on cucumber s e e d l i n g s i n the dark. A f t e r 17 hours i n the d a r k , the p l a n t s were exposed t o d a y l i g h t and they s u f f e r e d e x t e n s i v e photodynamic damage. The green l e a f y t i s s u e and the h y p o c o t y l became b l e a c h e d . I n b o t h c a s e s , the t i s s u e s s u f f e r e d a severe l o s s of t u r g i d i t y , p r o b a b l y due to the development of l e a k y c e l l memmembranes, f o l l o w e d by a r a p i d and severe d e h y d r a t i o n of the tissues. P r i o r t o l i g h t exposure, some of the s e e d l i n g s were anal y z e d and i n c r e a s e d c e l l u l a r l e v e l s of t o t a l t e t r a p y r r o l e s were found to be c o n c e n t r a t i o n dependent upon the sprayed d e l t a a m i n o l e v u l i n i c a c i d and 2 , 2 - d i p y r i d y l . When o t h e r p l a n t s were t r e a t e d s i m i l a r l y , i t became apparent t h a t the d e l t a - a m i n o l e v u l i n i c a c i d and 2 , 2 ' - d i p y r i d y l induced photodynamic a c t i o n c a u s i n g 3 d i f f e r e n t types of h e r b i c i d a l responses depending upon the t a r g e t s p e c i e s . The Type I r e s p o n s e , observed i n d i c o t s such as the cucumber, i s c h a r a c t e r i z e d by a c c u m u l a t i o n s of t e t r a p y r r o l e s i n l e a f y t i s s u e s , stems, and growing p o i n t s , and r a p i d death from photodynamic a c t i o n which i s d i r e c t l y dependent upon l i g h t i n t e n s i t y . The Type I I response i s observed i n o t h e r d i c o t s such as c o t t o n , k i d ney bean, and soybean. T e t r a p y r r o l e s are accumulated i n the l e a f y t i s s u e s , but not i n the stems. Leaves t h a t accumulate the t e t r a p y r r o l e s d i e v e r y r a p i d l y w i t h i n a few hours of l i g h t exposure, but the c o t y l e d o n s , stems, and growing p o i n t s remain u n a f f e c t e d . These p l a n t s c o u l d r e c o v e r from t h i s i n i t i a l damage by p r o d u c i n g new leaves. I t was a l s o observed t h a t , i f the p l a n t s were young enough 1

1

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

1.

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Photoactivated Compounds as Pesticides

15

t h a t the l e a v e s were e n c l o s e d by the c o t y l e d o n s , the p l a n t s were c o m p l e t e l y u n a f f e c t e d . The Type I I I response was e x h i b i t e d o n l y by monocotyledons, such as wheat, c o r n , o a t s , or b a r l e y . I n t h i s case, the p l a n t developed s m a l l n e c r o t i c r e g i o n s when the sprayed p l a n t was exposed t o l i g h t . The s e e d l i n g s grew v i g o r o u s l y and developed i n t o h e a l t h y p l a n t s . Rebeiz and h i s coworkers have thus developed the f i r s t photodynamic h e r b i c i d e . The p o p u l a r press has a l r e a d y g i v e n t h i s c l a s s o f h e r b i c i d e another name, " l a s e r h e r b i c i d e s . " By whatever name they w i l l be c a l l e d , these h e r b i c i d e s appear t o have a p r o m i s i n g f u t u r e . The d i f f e r e n t pathways of c h l o r o p h y l l b i o s y n t h e s i s s h o u l d a l l o w a degree of f l e x i b i l i t y so t h a t p r o d u c t s developed from t h i s c l a s s o f h e r b i c i d e s w i l l be t o l e r a n t t o the crop p l a n t s and t o x i c to the weeds. In 1986, Rebeiz and Hopen were awarded a patent c o v e r i n g the l a s e r h e r b i c i d e concept ( 1 2 2 ) . Miscellaneous Material There have been r e p o r t s of o t h e r m a t e r i a l s which may become import a n t as t h i s r e s e a r c h area d e v e l o p s . At t h i s t i m e , however, they have not a t t r a c t e d the a t t e n t i o n o f the p e s t i c i d e c l a s s e s t h a t were discussed e a r l i e r i n t h i s chapter. M a l t o t s y and F a b i a n (123-124) f i r s t found t h a t p o l y a r o m a t i c hydrocarbons were t o x i c to l a r v a e of the f r u i t f l y upon i r r a d i a t i o n w i t h UV l i g h t . The h i g h c a r c i n o g e n i c p o t e n t i a l of t h i s c l a s s of compounds has kept them from b e i n g e x p l o i t e d as much as would be expected i f t h e r e were no c a r c i n o g e n i c r i s k . Kagan and Kagan (125) addressed t h i s problem w i t h a comparative study the e f f e c t s of benzo[a]pyrene ( c a r c i n o g e n i c ) and pyrene ( n o n c a r c i n o g e n i c ) upon immature forms o f Aedes mosquitoes h e l d i n the dark or i r r a d i a t e d w i t h UV l i g h t . T h e i r r e s u l t s i n d i c a t e d t h a t c a r c i n o g e n i c i t y and p h o t o t o x i c i t y were not i n e x t r i c a b l y l i n k e d . L a t e r , Kagan e_t a l . (126) c a l l e d a t t e n t i o n t o the p o s s i b l e d e l e t e r i o u s e f f e c t s on a q u a t i c organisms of p o l y a r o m a t i c hydrocarbons i n a d v e r t a n t l y i n t r o duced i n t o the environment. Kagan e_t a l . (127) r e p o r t e d t h a t 2 , 5 - d i p h e n y l o x a z o l e , known t o workers i n s c i n t i l l a t i o n c o u n t i n g as POP, i s p h o t o t o x i c to the f i r s t i n s t a r of Aedes mosquito l a r v a e , t o c r u s t a c e a n s , and to the eggs o f f r u i t f l i e s . A s i m i l a r compound, 1 , 4 - b i s ( 5 - p h e n y l o x a z o l e 2-yl)benzene, known as POPOP, i s a l s o t o x i c , but t o a l e s s e r degree. Both can s e n s i t i z e the f o r m a t i o n of s i n g l e t oxygen. M o l e r o et^ a l . (128) r e p o r t e d a photodynamic a c t i v i t y i n r o o t t i s s u e mediated by b e r b e r i n e s u l f a t e and v i o l e t (420 nm) l i g h t . At low c o n c e n t r a t i o n s (nanomolar), r o o t growth i n h i b i t i o n was complete. The f i r s t p h o t o t o x i c l i g n a n , n o r d i h y d r o g u a i a r e t i c a c i d ( I X ) , from the l e a f r e s i n of the c r e o s o t e bush has been r e p o r t e d (Downum, K.R.; D o l e , J . ; R o d r i g u e z , E. Phytochem, i n p r e s s ) . Many more l i g n a n s are known, o c c u r r i n g i n many f a m i l i e s of p l a n t s , and they may become an i m p o r t a n t f u t u r e source of p h o t o c h e m i c a l l y a c t i v e chemicals.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

LIGHT-ACTIVATED PESTICIDES

OH

OH

0

OH

0

OH

Hypericin

OH

0

OH

0

Cercosporin

Structure VIII

Nordihydroguaiaretic acid

S t r u c t u r e IX

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

1. HEITZ

Photoactivated Compounds as Pesticides

17

Conclusions It i s apparent that the concept of l i g h t activation of molecules to enhance b i o l o g i c a l a c t i v i t y i s a concept which i s both intriguing and currently available- Although there are applications which would not allow catalysis by l i g h t , such as the photonegative insects and most root tissue i n plants, there is a wide and diverse population of pests which do function i n the l i g h t . The f i r s t tentative steps torwards application have been taken using available synthetic chemicals and known plant materials, a l l of which were i d e n t i f i e d through general screening programs. It i s to be hoped that the next steps may follow at least i n part the approaches of Constantin Rebeiz and his coworkers i n which the toxic molecule was designed from known p r i c i p l e s of the biochemistry of the plant target. In fact, although there are no known examples thus f a r , i t would appear that the general area of photoaffinity l a b e l l i n g for the development of within the pest are more completely understood. Other fundamental areas of l i g h t activation systems may s i m i l a r l y be future watersheds for pesticides based on this approach. Acknowledgments This work was supported i n f u l l by the M i s s i s s i p p i A g r i c u l t u r a l and Forestry Experiment Station. The author would l i k e to thank Mrs. Debbie Smith and Mrs. Ann Smithson for their assistance i n typing the manuscript. MAFES publication number 6524. Literature Cited 1. Heitz, J.R. In Insecticide Mode of Action; Coats, J . R . , Ed.; Academic: New York, 1982; pp. 429-457. 2. Robinson, J.R. Res. Rev. 1983, 88, 69-100. 3. Arnason, T . ; Towers, G.H.N.; Philogene, B.J.R.; Lambert, J.D.H. In Plant Resistance to Insects; Hedin, P.A., Ed.; ACS Symposium Series No. 208; American Chemical Society, Washington, DC, 1983; pp. 139-151. 4. Rebeiz, C.A.; Montazer-Zouhoor, A.; Hopen, H . J . ; Wu, S.M. Enzyme Microb. Technol. 1984, 6, 390-401. 5. Towers, G.H.N. Can. J . Bot. 1984, 62, 2900-2911. 6. Cooper, G.K.; Nitsche, C.I. Bioorg. Chem. 1985, 13, 362-374. 7. Knox, J . P . ; Dodge, A.D. Phytochem. 1985, 24, 889-896. 8. Downum, K.R. In Natural Resistance of Plants to Pests:Roles of Allelochemicals; Green, M.B.; Hedin, P.A., Eds.; ACS Symposium Series No. 296; American Chemical Society, Washington, DC, 1986; pp. 197-205. 9. Marcacci, A. Arch. Ital. Biol. 1888, 9, 2. 10. Rabb, O. Z. fur Biol. 1900, 39, 524-546. 11. Jodlbauer, A . ; von Tappeiner, H. Muench. Med. Wochenschr. 1904, 26, 1139-1141. 12. Spikes, J . D . ; Glad, B.W. Photochem. Photobiol. 1964, 3, 471-487. 13. Edwards, W.F. Text. World. 1921, 60, 1111-1113. 14. Chang, H.T. Mosquito News. 1946, 6, 122-125.

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David, J . C.R. Acad. Sci. Paris. 1955, 241, 116-118. David, J . Bull. Biol. France Belgique. 1963, 97, 515-530. Zacharuk, R.Y. Can. J . Zool. 1963, 41, 991-996. Gangwere, S.K.; Chavin, W.; Evans, F.C. Annal. of Entomol. Soc. Amer. 1964, 57, 662-669. Kolyer, J.M. J . Res. Lep. 1966, 5, 136-152. Peters, T.M.; Chevone, B.I. Mosquito News. 1968, 28, 24-28. Daum, R . J . ; Gast, R.T.; Davitch, T.B. J . Econ. Entomol. 1969, 62, 943. Hayes, D.K.; Schechter, M.S. J . Econ. Entomol. 1070, 63, 997. Barbosa, P.; Peters, T.M. J . Med. Entomol. 1970, 7, 693-696. Barbosa, P.; Peters, T.M. Histochem. J . 1971, 3, 71. Hendricks, D.E. J . Econ. Entomol. 1971, 64, 1404. Jones, R . L . ; Harrell, E.A.; Snow, J.W. J . Econ. Ent. 1972, 65, 123-126. Bridges, A . C . ; Cocke, J.; Olson, J . K . ; Mayer, R.T. Mosquito News. 1977, 37, 227 Barbieri, A. Riv. Malariol Schildmacher, H. Biol. Zentralbl. 1950, 69, 468-477. Ware, G.W. Pesticides Theory and Application; W.H. Freeman: San Francisco, 1978; p. 12. Blum, H.F. Photodynamic Action and Diseases Caused by Light; Rheinhold: New York, 1941. Spikes, J . D . ; Straight, R. Annu. Rev. Phys. Chem. 1967, 18, 409-436. Spikes, J . D . ; Livingston, R. Adv. Radiat. Biol. 1969, 3, 29121. Grossweiner, L . I . Photophysiology, 1970, 5, 1-33. Wilson, T . ; Hastings, J.W. Photophysiology, 1970, 5, 49-95. Krinsky, N.I. Trends Biochem. Sci. (Pers. Ed.), 1977, 2, 35-38. Spikes, J.D. In The Science of Photobiology; Smith, K . C . , Ed.; Plenum, New York, 1977; p. 87-112. Yoho, T.P.; Butler, L . ; Weaver, J . E . J . Econ. Entomol. 1971, 64, 972-973. Yoho, T.P. Ph.D. Dissertation, West Virginia University, Morgantown, 1972. Yoho, T.P.; Weaver, J.E.; Butler, L. Environ, Entomol. 1973, 2, 1092-1096. Yoho, T.P.; Butler, L . ; Weaver, J . E . Environ. Entomol. 1976, 5, 203-204. Graham, K.; Wrangler, E . ; Aasen, L.H. Can. J. Zool. 1972, 50, 1625-1629. Broome, J . R . ; Callaham, M.F.; Lewis, L.A.; Ladner, C.M.; Heitz, J.R. Comp. Biochem. Physiol. 1975, 51C, 117-121. Broome, J . R . ; Callaham, M.F.; Heitz, J.R. Environ. Entomol. 1975a, 4, 883-886. Callaham, M.F.; Broome, J . R . ; Lindig, O.H.; Heitz, J.R. Environ. Entomol. 1975, 4, 837-841. Fondren, J.E., Jr.; Heitz, J.R. Environ. Entomol. 1978, 7, 843-846. Fondren, J . E , Jr.; Norment, B.R.; Heitz, J.R. Environ. Entomol. 1978, 7, 205-208. David, R.M.; Heitz, J.R. J . Agr. Food Chem. 1978, 26, 99-101. Callaham, M.F.; Lewis, L . A . ; Holloman, M.E.; Broome, J.R.; Heitz, J.R. Comp. Biochem. Physiol. 1975a, 51C, 123-128.

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50. Callaham, M.F.; Palmertree, C.O.; Broome, J.R.; Heitz, J.R. Pest. Biochem. Physiol. 1977, 7, 21-27. 51. Weaver, J.E.; Butler, L.; Yoho, T.P. Environ. Entomol. 1976, 5, 840. 52. Weaver, J . E . ; Butler, L.; Amrine, J.W., Jr. Environ. Entomol. 1982, 11, 463-466. 53. Broome, J.R.; Callaham, M.F.; Poe, W.E.; Heitz, J.R. Chem.Biol. Interact. 1976, 14, 203-206. 54. Callaham, M.F.; Broome, J.R.; Poe, W.E.; Heitz, J.R. Environ. Entomol. 1977a, 6, 669-673. 55. Fondren, J.E., Jr.; Heitz, J.R. Environ. Entomol. 1978a, 7, 891-894. 56. Fondren, J.E., Jr.; Heitz, J.R. Environ. Entomol. 1979, 8, 432-436. 57. Lavialle, M.; Dumortier, B. C.R. Hebd. Seances Acad. Sci. 1978, 287, 875-878. 58. Clement, S.L.; Schmidt J. Econ. Entomol. 1980 59. Creighton, C.S.; McFadden, T . L . ; Schalk, J.M. J . Georgia Entomol. Soc. 1980, 15, 66-68. 60. Pimprikar, G.D.; Norment, B.R.; Heitz, J.R. Environ. Entomol. 1979, 9, 856-859. 61. Pimprikar, G.D.; Fondren, J.E., Jr.; Heitz, J.R. Environ. Entomol. 1980a, 9, 53-58. 62. Pimprikar, G.D.; Noe, B . L . ; Norment, B.R.; Heitz, J.R. Environ. Entomol. 1980b, 9, 785-788. 63. Carpenter, T . L . ; Heitz, J.R. Environ. Entomol. 1980, 9, 533537. 64. Carpenter, T . L . ; Heitz, J.R. Environ. Entomol. 1981, 10, 972-976. 65. Fairbrother, T . E . ; Essig, H.W.; Combs, R . L . ; Heitz, J.R. Environ. Entomol. 1981, 10, 506-510. 66. Carpenter, T . L . ; Mundie, T . G . ; Ross, J . H . ; Heitz, J.R. Environ. Entomol. 1981, 10, 953-955. 67. Crounse, N.; Heitz, J.R. U.S. Patent 4 320 140, 1982. 68. Carpenter, T . L . ; Johnson, L . H . ; Mundie, T.G.; Heitz, J.R. J. Econ. Entomol. 1984, 77, 308-312. 69. Sakurai, H . ; Heitz, J.R. Environ. Entomol. 1982, 11, 467-470. 70. Respicio, N. C., Heitz, J.R. J . Econ. Entomol. 1983, 76, 1005-1008. 71. Respicio, N.C.; Heitz, J.R. J . Econ. Entomol. 1986, 79, 315317. 72. Pimprikar, G.D.; Fondren, J.E., Jr.; Greer, D.S.; Heitz, J.R. Southwest. Entomol. 1984, 9, 218-222. 73. Pimprikar, G.D.; Heitz, J.R. J . Miss. Acad. Sci. 1984, 29, 77-80. 74. Carpenter, T . L . ; Respicio, N.C.; Heitz, J.R. Environ. Entomol. 1984a, 13, 1366-1370. 75. Respicio, N.C.; Carpenter, T . L . ; Heitz, J.R. J . Econ. Entomol. 1985, 78, 30-34. 76. Carpenter, T . L . ; Respicio, N.C.; Heitz, J.R. J . Econ. Entomol. 1985, 78, 232-237. 77. Knox, J . P . : Dodge, A.D. Planta, 1985, 164, 22-29. 78. Knox, J . P . ; Dodge, A.D. Planta, 1985a, 164, 30-34.

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20

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79. Robinson, J.R.; Beatson, E.P. Pest. Biochem. Physiol. 1985, 24, 375-383. 80. Hawkins, J . A . ; Healey, M.C.; Johnson-Delivorias, M.H.; Heitz, J.R. Veterin. Parasitol. 1984, 16, 35-41. 81. Berenbaum, M. J . Chem. Ecol. 1986, 12, 807-948. 82. Ivie, G.W. In Effects of Poisonous Plants on Livestock; Keeler, R., Van Kampen, K . , James, L., Eds.; Academic: New York, 1978; pp. 475-485. 83. Berenbaum, M. Science, 1978, 201, 532-534. 84. Scott, B.R.; Pathak, M.A.; Mohn, G.R. Mutat. Res. 1976, 39, 29-74. 85. Song, P.-S.; Tapley, K.J., Jr. Photochem. Photobiol. 1979, 29, 1177-1197. 86. Berenbaum, M.; Feeny, P. Science, 1981, 212, 927-929. 87. Ivie, G.W.; Bull, D.L.; Beier, R.C.; Pryor, N.W.; Oertli, E.H. Science, 1983, 221, 374-376. 88. Bull, D.L.; Ivie, G.W. J. Chem. Ecol. 1984 89. Ivie, G.W.; Bull, D.L.; Beier, R.C.; Pryor, N.W. J . Chem Ecol. 1986, 12, 869-882. 90. Ashwood-Smith, M . J . ; Ring, R.A.; Liu, M.; Phillips, S.; Wilson, M. Can. J. Zool. 1984, 62, 1971-1976. 91. Bull, D . L . ; Ivie, G.W.; Beier, R.C.; Pryor, N.W. J . Chem. Ecol. 1986, 12, 883-890. 92. Uhlenbroek, J . H . ; Bijloo, J.D. Rec. Trav. Chim. Pays-Bas Belg. 1958, 77, 1004-1008. 93. Gommers, F . J . Nematologica, 1972, 18, 458-462. 94. Gommers, F.J.; Geerligs, J.W.G. Nematologica, 1973, 19, 389-393. 95. Bakker, J.; Gommers, F.J.; Nieuwenhuis, I.; Wynberg, H. J. Biol. Chem. 1979, 254, 1841-1844. 96. Gommers, F.J.; Bakker, J.; Smits, L. Nematologica, 1980, 26, 369-375. 97. Wat, C.-K.; Prasad, S.K.; Graham, E.A.; Partington, S.; Arnason, T.; Towers, G.H.N. Biochem. Syst. and Ecol. 1981, 9, 59-62. 98. Arnason, T . ; Swain, T.; Wat, C.-K.; Graham, E . A . ; Partington, S.; Towers, G.H.N.; Lam, J. Biochem. Syst. and Ecol. 1981, 9, 63-68. 99. Arnason, T.; Chan, G.F.Q.; Wat, C.K.; Downum, K.; Yamamoto, E.; Towers, G.H.N. Photochem. Photobiol. 1981a, 33, 821-824. 100. McLachlan, D.; Arnason, T . ; Lam, J. Biochem. Syst. and Ecol. 1986, 14, 17-23. 101. Arnason, T.; Swain, T.; Wat, C.K.; Graham, E . A . ; Partington, S.; Tow, G.H.N.; Lam, J. Biochem. Syst. and Ecol. 1981b, 9, 63-68. 102. Kagan, J.; Chan, G Experientia, 1983, 39, 402-403. 103. Kagan, J.; Prakash, I.; Dhawan, S.N.; Jaworski, J.A. Photobiochem. Photobiophys. 1984, 8, 25-33. 104. Kagan, J.; Kolyvas, C.P.; Lam, J. Experientia, 1984a, 40, 1396-1397. 105. Downum, K.R.; Rosenthal, G.A.; Towers, G.H.N. Pest. Biochem. Physiol. 1984, 22, 104-109. 106. Kagan, J.; Hasson, M.; Grynspan, F. Biochim. Biophys. Acta, 1984b, 802, 442-447.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

1. HEITZ

Photoactivated Compounds as Pesticides

21

107. Reyftmann, J . P . ; Kagan, J.; Santus, R.; Morliere, P. Photochem. Photobiol. 1985, 41, 1-7. 108. Kagan, J.; Kagan, E.D.; Siegneurie, E. Chemosphere, 1986, 15, 49-57. 109. Towers, G.H.N.; Arnason, J . T . ; Wat, C.K.; Lambert, J.D.H. Can Pat. 1,173,743, 1984. 110. Horsley, C.H.J. Pharmacol. 1934, 50, 310-322. 111. Brockmann, H.H. Prog. Org. Chem. 1952, 1, 64-82. 112. Brockmann, H.H. Proc. Chem. Soc. London, 1957, 304-312. 113. Yamazaki, S.; Okube, A.; Akiyama, Y.; Fuwa, K. Agricult. Biol. Chem. 1975, 39, 287-288. 114. Macri, F . ; Vianello, A. Plant Cell and Environ. 1979, 2, 267-271. 115. Daub, M.E. Phytopathology, 1982, 72, 370-374. 116. Daub, M.E. Plant Physiol. 1982a, 69, 1361-1364. 117. Daub, M.E.; Briggs, S.P. Plant Physiol. 1983, 71, 763-766. 118. Daub, M.E.; Hangarter 119. Knox, J.P; Dodge, 19-25. 120. Ellsworth, R.K.; Aronoff, S. Arch. Biochem. Biophys. 1969, 130, 374-383. 121. Rebeiz, C.A. Chemtech. 1982, 12, 52-63. 122. Rebeiz, C.A.; Hopen, H.J. PCT Int. Appl. WO 8, 600, 785. 123. Maltotsy, A . G . ; Fabian, G. Nature, 1946, 149, 877. 124. Maltotsy, A.G.; Fabian, G. Arch. Biol. Hungarica, 1947, 17, 165-170. 125. Kagan, J.; Kagan, E. Chemosphere, 1986a, 15, 243-251. 126. Kagan, J.; Kagan, E.D.; Kagan, I.A.; Kagan, P.A.; Quigley, S. Chemosphere, 1985, 14, 1829-34. 127. Kagan, J.; Kolyvas, C.P.; Jaworski, J . A . ; Kagan, E.D.; Kagan, I.A.; Zang, L . - H . Photochem. Photobiol. 1984c, 40, 479-483. 128. Molero, M.L.; Hazen, M . J . ; Stockert, J.C. J . Plant Physiol. 1985, 120, 91-94. RECEIVED November 20, 1986

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 2

Type I and Type II Mechanisms of Photodynamic Action Christopher S. Foote Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90024

Mechanisms of photooxidatio discussed, and methods of determining photooxidation mechanisms reviewed. Two cases that have been particularly well studied, cercosporin and a-terthienyl, are used to exemplify the techniques.

M a n y c h e m i c a l s , i n c l u d i n g natural c e l l c o n s t i t u e n t s , c a n a b s o r b light a n d p h o t o s e n s i t i z e d a m a g e to o r g a n i s m s . S o m e of t h e s e c o m p o u n d s a r e u s e d by o r g a n i s m s (including man) to attack o r d e f e n d a g a i n s t other o r g a n i s m s . T h i s p r o c e s s , c a l l e d " p h o t o d y n a m i c a c t i o n " , requires o x y g e n a n d d a m a g e s biological target m o l e c u l e s by photosensitized oxidation. B i o c h e m i c a l effects i n c l u d e e n z y m e d e a c t i v a t i o n (through d e s t r u c t i o n of s p e c i f i c a m i n o a c i d s , particularly methionine, histidine, a n d tryptophan), nucleic acid o x i d a t i o n (primarily of g u a n i n e ) , a n d m e m b r a n e d a m a g e (by o x i d a t i o n of unsaturated fatty a c i d s a n d cholesterol) (1. 2). M e c h a n i s m s of P h o t o o x y g e n a t i o n P h o t o s e n s i t i z e d oxidations are initiated by absorption of light by a sensitizer, w h i c h c a n be a d y e o r pigment, a ketone o r q u i n o n e , a n a r o m a t i c m o l e c u l e , or m a n y other t y p e s of c o m p o u n d . T h e s e n s i t i z e r ( S e n s ) is c o n v e r t e d to a n electronically e x c i t e d state by absorption of a photon. T h e initial product is a s h o r t - l i v e d s i n g l e t ( S e n s ) ; in m a n y c a s e s , this u n d e r g o e s i n t e r s y s t e m c r o s s i n g to t h e longer-lived triplet ( S e n s ) . B e c a u s e t h e singlet g e n e r a l l y h a s a very short lifetime, only reactants at relatively high c o n c e n t r a t i o n c a n interact with it before it d e c a y s ; h o w e v e r , m u c h l o w e r c o n c e n t r a t i o n s a r e sufficient to react with the longer-lived triplet state. 1

3

Sens

>

1

Sens

>

3

Sens

hv 0097-6156/87/0339-0022S06.00/0 © 1987 American Chemical Society

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

2.

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Mechanisms of Photodynamic Action

23

T h e r e are two m e c h a n i s m s of p h o t o s e n s i t i z e d oxidation, n a m e d " T y p e I" a n d " T y p e II" by G o l l n i c k (3) ( s e e F i g . 1). In the T y p e I p r o c e s s , substrate or s o l v e n t r e a c t s with the s e n s i t i z e r e x c i t e d s t a t e (either singlet or triplet, S e n s * ) to g i v e r a d i c a l s or radical i o n s , r e s p e c t i v e l y , by h y d r o g e n a t o m or electron transfer. R e a c t i o n of t h e s e r a d i c a l s with o x y g e n g i v e s o x y g e n a t e d products. In the T y p e II p r o c e s s , the e x c i t e d s e n s i t i z e r reacts with o x y g e n to form singlet m o l e c u l a r o x y g e n ( 0 2 ) , w h i c h t h e n r e a c t s with s u b s t r a t e to 1

form t h e p r o d u c t s . T h e T y p e I a n d T y p e II m e c h a n i s m s a r e a l w a y s in competition; factors which govern the competition include o x y g e n c o n c e n t r a t i o n , the reactivities of the substrate a n d of the s e n s i t i z e r e x c i t e d state, the substrate concentration, a n d the singlet o x y g e n lifetime (4). T h e s e factors will be d i s c u s s e d in more detail in a s u b s e q u e n t s e c t i o n . Sens Type I Radicals •

<

h V

II

Sens* Substrate

°2

I

Substrate

| °

2

So.vent

Oxygenated Products

T r i

P

l e t

Oxygenated Products

Fig. 1. Mechanisms of Photosensitized Oxidation

Type i Prppesses E l e c t r o n transfer both to a n d from m o l e c u l e s t a k e s p l a c e m o r e readily in the e x c i t e d state than in the g r o u n d state (5.6). T h i s follows from the fact that a n e l e c t r o n is p r o m o t e d from a strongly b i n d i n g orbital to o n e that is l e s s strongly binding o n g o i n g from the g r o u n d state to the e x c i t e d state. T h i s p r o c e s s results in the production of a r e d u c i n g e l e c t r o n a n d a n o x i d i z i n g hole in the excited state, a s s h o w n in F i g . 2. A w e l l - s t u d i e d e x a m p l e of the e l e c t r o n - t r a n s f e r T y p e I p r o c e s s is the oxidation of a r o m a t i c olefins (Donor), by e l e c t r o n - p o o r a r o m a t i c s s u c h a s d i c y a n o a n t h r a c e n e ( D C A ) , w h i c h results in transfer of a n e l e c t r o n to the singlet excited state of the a r o m a t i c from the olefin (7). T h e resulting radical a n i o n of the s e n s i t i z e r is i m m e d i a t e l y r e o x i d i z e d by o x y g e n , p r o d u c i n g a s u p e r o x i d e i o n - a r o m a t i c r a d i c a l c a t i o n p a i r . T h e s e r e a c t to g i v e the o b s e r v e d products, mainly oxidative c l e a v a g e of the olefins.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

24

LIGHT-ACTIVATED PESTICIDES

Empty Orbitals

Weakly B o u n d Electron (Reducing

Empty Hole ( O x i d i z ng) Filled Orbitals

Ground State

Excited State

F i g . 2 . Electron Promotion in Excited State

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

2.

25

Mechanisms of Photodynamic Action

FOOTE

DCA

D C A + Donor-

+ Donor

•>

<>2 +

D0

2

DCA

T h e T y p e I reaction c a n a l s o result in h y d r o g e n abstraction, giving radical p r o d u c t s . T h e s e r a d i c a l s c a n react directly with o x y g e n to g i v e p e r o x i d e s or initiate r a d i c a l c h a i n a u t o x i d a t i o n . H y d r o g e n a b s t r a c t i o n is particularly c o m m o n with k e t o n e a n d q u i n o n e s e n s i t i z e r s , but a l s o o c c u r s with m a n y d y e s , although usually les d o n o r s promote this reactio R C=0 2

+

R'-H

R C-0H o

+

R'«

Radical Chain Reactions T h e T y p e II

Process

T h e T y p e II r e a c t i o n p r o d u c e s s i n g l e t m o l e c u l a r o x y g e n , w h i c h r e a c t s directly with s u b s t r a t e s to give o x y g e n a t e d products or d e c a y s to the g r o u n d state if it fails to react. T h e rate of d e c a y is strongly d e p e n d e n t on solvent: in w a t e r , the lifetime of singlet o x y g e n is a b o u t four m i c r o s e c o n d s , while in o r g a n i c s o l v e n t s (and p r e s u m a b l y a l s o in the lipid r e g i o n s of m e m b r a n e s ) , the lifetime is o n the order of ten to twenty t i m e s longer (8.9).

3

°2

*

1

°2

Substrate »

Substrate •

0

2

T w o major c l a s s e s of singlet o x y g e n r e a c t i o n s are additions to olefins with allylic h y d r o g e n s , giving allylic h y d r o p e r o x i d e s , with a shift in position of the d o u b l e b o n d (the " e n e " reaction, 1Q), a n d addition to d i e n e s , a r o m a t i c s , a n d h e t e r o c y c l e s , giving e n d o p e r o x i d e s (the D i e l s - A l d e r reaction, H ) .

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

LIGHT-ACTIVATED PESTICIDES

26

O t h e r p r o c e s s e s include reaction of electron-rich olefins to give unstable f o u r - m e m b e r e d ring p e r o x i d e s c a l l e d d i o x e t a n e s ( 1 £ ) , oxidation of s u l f i d e s to s u l f o x i d e s ( v i a a n i n t e r m e d i a t e " p e r s u l f o x i d e " , 12), a n d r e a c t i o n of electron-rich p h e n o l s , including tocopherol, to unstable h y d r o p e r o x y d i e n o n e s (14).

1

0

2

+ R S

> R S+00-

2

2

>

2R S+0 2

M a n y c o m p o u n d s d e a c t i v a t e (i. e . q u e n c h ) singlet o x y g e n efficiently without reacting (15.). F o r e x a m p l e , p - c a r o t e n e inhibits p h o t o o x i d a t i o n of m a n y c o m p o u n d s efficiently at e v e n very l o w c o n c e n t r a t i o n s b y a n e n e r g y t r a n s f e r m e c h a n i s m , w i t h o u t b e i n g a p p r e c i a b l y o x i d i z e d itself. O t h e r c o m p o u n d s s u c h a s D A B C O (1,4-diazabicyclooctane) a n d a z i d e i o n q u e n c h singlet o x y g e n b y a c h a r g e - t r a n s f e r p r o c e s s . P h e n o l s a n d s u l f i d e s a l s o q u e n c h singlet o x y g e n , in competition with their oxidation. F o r e x a m p l e , cct o c o p h e r o l q u e n c h e s singlet o x y g e n at a high rate in all s o l v e n t s , but reacts rapidly only in protic s o l v e n t s (16-18). f

1

1

0

2

+ DABCO

0

2

+ Car

>

3Car

>0 -— D A B C O + 2

+

3

0

>

2

DABCO

+ 3(>

2

S i n g l e t o x y g e n i s a n electronically e x c i t e d m o l e c u l e , a n d c a n return to the g r o u n d state with e m i s s i o n of light ( I S ) . T h e r e a r e t w o t y p e s of singlet o x y g e n l u m i n e s c e n c e , from a single m o l e c u l e at 1.27 p.m, a n d d i m o l " lumin e s c e n c e at 6 3 4 a n d 7 0 4 n m . Both t y p e s of l u m i n e s c e n c e a r e very inefficient b e c a u s e t h e lifetime of singlet o x y g e n in solution is short c o m p a r e d to t h e radiative lifetime. B e c a u s e t h e d i m o l e m i s s i o n d e p e n d s o n a b i m o l e c u l a r collision b e t w e e n t w o short-lived s p e c i e s , its efficiency a l s o d e p e n d s o n the concentration of singlet o x y g e n . H

1

0

2

> hv (1.27n)

2(1Q ) 2

> hv

(634,704nm)

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Mechanisms of Photodynamic Action

Determination of

Mechanism

A s m e n t i o n e d in the introduction, high s e n s i t i z e r reactivity, high s u b s t r a t e reactivity a n d c o n c e n t r a t i o n , low o x y g e n c o n c e n t r a t i o n , a n d short singlet o x y g e n lifetimes favor the T y p e I m e c h a n i s m , while the opposite factors favor the T y p e II. O n e of the m o s t direct m e t h o d s of d e t e r m i n i n g w h e t h e r a r e a c t i o n is p r o c e e d i n g v i a a T y p e I or a T y p e II m e c h a n i s m is to vary s u b s t r a t e a n d o x y g e n c o n c e n t r a t i o n a n d determine the a m o u n t of products f o r m e d u n d e r v a r i o u s c o n d i t i o n s . T h i s t e c h n i q u e is particularly u s e f u l in h o m o g e n e o u s s o l u t i o n , e s p e c i a l l y w h e r e there are distinct s e t s of p r o d u c t s from the two m e c h a n i s m s . At sufficiently high o x y g e n a n d / o r low s u b s t r a t e c o n c e n t r a t i o n , a r e a c t i o n c a n b e f o r c e d into a c l e a n T y p e II p a t h w a y , w h e r e a s the T y p e I p a t h w a y c a n b e f o r c e d u n d e r the o p p o s i t e c o n d i t i o n s . C h a n g i n g solvent to o n e in w h i c h the singlet o x y g e n lifetime is longer helps to f a v o r the T y p e II m e c h a n i s m m e m b r a n e , p r o t e i n , or n u c l e i o r g a n i s m s , a n d t e n d s to favor T y p e I m e c h a n i s m s b e c a u s e of the effective i n c r e a s e in substrate concentration (20.21). T h e r e are two e x a m p l e s w h e r e the competition b e t w e e n T y p e I a n d T y p e II m e c h a n i s m s has been particularly well d o c u m e n t e d , 1,1d i p h e n y l m e t h o x y e t h y l e n e ( D P M E , 22) a n d d i m e t h y l s t i l b e n e (£3). In both c a s e s , the reaction c a n be m a n i p u l a t e d by m e a n s of the factors d e s c r i b e d a b o v e to give d i o x e t a n e products v i a the electron-transfer p a t h w a y or DielsA l d e r or e n e products, respectively, v i a the T y p e II route.

O

Rearrangement Products

Oxygen DPME

OCH

3

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

28

LIGHT-ACTIVATED PESTICIDES

E s t a b l i s h i n g the m e c h a n i s m of p h o t o s e n s i t i z e d o x i d a t i o n s in c o m p l e x s y s t e m s is a difficult t a s k ( 2 4 - 2 6 ) . Kinetic tests a n d the u s e of inhibitors for v a r i o u s r e a c t i v e s p e c i e s a r e m o r e a m b i g u o u s t h a n in h o m o g e n e o u s s o l u t i o n , b e c a u s e r e a g e n t s a r e often c o m p a r t m e n t a l i z e d , b o u n d , or l o c a l i z e d , a n d it is rarely p o s s i b l e to k n o w the local c o n c e n t r a t i o n s of v a r i o u s reacting s p e c i e s , s e n s i t i z e r s , q u e n c h e r s , a n d traps. M a n y w o r k e r s h a v e u s e d a l l e g e d l y s p e c i f i c t r a p s or q u e n c h e r s for v a r i o u s reactive s p e c i e s , including singlet o x y g e n , s u p e r o x i d e ion ( 0 - ) , 2

hydroxyl radical (OH-), peroxy radicals ( R O O - ) , a n d other oxidants. H o w e v e r , the specificity of traps a n d inhibitors for oxidants requires far more s t u d y t h a n it h a s r e c e i v e d . F o r i n s t a n c e , all r e a g e n t s a n d q u e n c h e r s for singlet o x y g e n h a v e low oxidation potentials a n d will a l s o interact with other oxidants. A l s o , almost all q u e n c h e r s of singlet o x y g e n c a n q u e n c h s e n s i t i z e r e x c i t e d s t a t e s a s w e l l . Q u e n c h i n g of s e n s i t i z e r e x c i t e d s t a t e s c a n be d i s t i n g u i s h e d from singlet o x y g e n q u e n c h i n g by d e t e r m i n i n g the d e g r e e of inhibition at s e v e r a l o x y g e n c o n c e n t r a t i o n s , s i n c e if singlet o x y g e n is being q u e n c h e d , t h e d e g r e e of i n h i b i t i o n will not d e p e n d o n t h e o x y g e n concentration. Interconversions a n d interactions a m o n g reactive s p e c i e s c o m p l i c a t e the p r o c e s s further. In both T y p e I a n d T y p e II reactions, the initial products are often p e r o x i d e s , w h i c h c a n b r e a k d o w n to i n d u c e free r a d i c a l r e a c t i o n s . S u c h s e c o n d a r y thermal reactions h a v e b e e n s h o w n to c a u s e m u c h of the p h o t o d y n a m i c d a m a g e o b s e r v e d in m e m b r a n e s u n d e r s o m e c o n d i t i o n s ( 2 7 . 2 8 ) . R a d i c a l c h a i n s c a n c a u s e the o x i d a t i o n of m a n y m o l e c u l e s of

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Mechanisms of Photodynamic Action

starting material for e a c h primary product. F o r this r e a s o n , product a n a l y s i s m a y reflect m a i n l y s e c o n d a r y c h a i n p r o c e s s e s rather t h a n the primary reaction m e c h a n i s m . S i m i l a r c o m m e n t s apply to inhibition s t u d i e s . M e t h o d s of a s s e s s i n g the relative i m p o r t a n c e of v a r i o u s p r o c e s s e s are n e e d e d . D e t e c t i o n of a n i n t e r m e d i a t e is a n e c e s s a r y but not sufficient c o n d i t i o n for its h a v i n g a c a u s a t i v e role in a p r o c e s s . It d o e s little g o o d to s h o w that a reactive intermediate is present without b e i n g a b l e to estimate what fraction of the overall oxidation it c a u s e s . S u c h quantitation h a s rarely b e e n a c c o m p l i s h e d in h e t e r o g e n e o u s s y s t e m s . T e c h n i q u e s for

Characterizing Singlet

Oxygen

A large n u m b e r of t e c h n i q u e s h a v e b e e n d e v e l o p e d for detection of p o s s i b l e reactive intermediates in biological o x y g e n d a m a g e ( 2 4 . 29). F o r r e a s o n s of s p a c e , this report will c o n c e n t r a t detection a n d characterizatio C h e m i c a l T r a p s . A large n u m b e r of c o m p o u n d s h a v e b e e n a d d e d to r e a c t i n g s y s t e m s a s t r a p s for singlet o x y g e n , a n d t h e f o r m a t i o n of the s u p p o s e d l y characteristic products u s e d a s a n indication of the intermediacy of 0 2 - F o r e x a m p l e , dimethylfuran reacts with singlet o x y g e n to give the 1

d i k e t o n e s h o w n b e l o w a s the ultimate product. Unfortunately, s o d o a very large n u m b e r of other oxidants. In fact, furans are a prime e x a m p l e of very n o n s p e c i f i c singlet o x y g e n traps (24)-

A d i a g n o s t i c t r a p for s i n g l e t o x y g e n is c h o l e s t e r o l , w h i c h r e a c t s with singlet o x y g e n to g i v e the 5-cc h y d r o p e r o x i d e ; r e a c t i o n s with r a d i c a l a n d other o x i d a n t s g i v e c o m p l e x mixtures, but the 5-cc product is not a m o n g t h e m (20). T h i s s y s t e m is s o m e w h a t limited b e c a u s e of the low reactivity of c h o l e s t e r o l with singlet o x y g e n . A l t h o u g h c h o l e s t e r o l is not s o l u b l e in water, it c a n be b o u n d to m i c r o s p h e r e s , allowing its u s e in a q u e o u s s y s t e m s (31). R

HO

HO OOH

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

30

LIGHT-ACTIVATED PESTICIDES

A s e c o n d t r a p p i n g s y s t e m w h i c h a l s o a p p e a r s to b e s p e c i f i c u s e s s u i t a b l y s u b s t i t u t e d a n t h r a c e n e s (32. 3 3 ) . A n t h r a c e n e d e r i v a t i v e s a r e c o n s i d e r a b l y m o r e reactive t h a n c h o l e s t e r o l . T h e s e c o m p o u n d s c a n b e m a d e s o l u b l e in a n y m e d i u m by s u i t a b l e c h o i c e of s u b s t i t u e n t s . O n e d r a w b a c k to this s y s t e m is that a n t h r a c e n e s are a l s o p h o t o s e n s i t i z e r s , s o that w h e n s m a l l a m o u n t s of product are f o r m e d , adventitious photooxidation must be carefully ruled out.

A third trapping s y s t e m m a k e s u s e of the fact that p o l y u n s a t u r a t e d fatty a c i d s r e a c t with s i n g l e t o x y g e n to g i v e a mixture of c o n j u g a t e d a n d u n c o n j u g a t e d i s o m e r s of the product h y d r o p e r o x i d e s , w h e r e a s only the c o n j u g a t e d i s o m e r s are f o r m e d o n radical attack (34). T h e u n c o n j u g a t e d p r o d u c t s t h u s s e r v e a s c h a r a c t e r i s t i c s i n g l e t o x y g e n fingerprints. This s y s t e m , like the c h o l e s t e r o l trap, is s o m e w h a t difficult to u s e , s i n c e the i s o m e r s must be s e p a r a t e d by H P L C .

A f u r t h e r s y s t e m is s u g g e s t e d b y C a d e t , w h o h a s i s o l a t e d t h e h y d r o x y l a c t a m s h o w n b e l o w f r o m p h o t o o x i d a t i o n of g u a n o s i n e , a n d h a s s h o w n that this c o m p o u n d c a n be u s e d a s a fingerprint for the p r e s e n c e of s i n g l e t o x y g e n (35.). T h i s c o m p o u n d i s p r o b a b l y t h e p r o d u c t of r e a r r a n g e m e n t of t h e initial p e r o x i d e , w h i c h is not s t a b l e at r o o m temperature.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

31

Mechanisms of Photodynamic Action

2. FOOTE

Two other trapping systems are used primarily for kinetic characterization of singlet oxygen; neither is likely to be useful in systems where there is more than one strong oxidant. One is a sensitive system using the production and ESR detection of the nitroxide radical from a tertiary amine (a process whose mechanism and stoichiometry are poorly understood) ( 3 6 ) . The second uses the bleaching of a p-nitrosodimethylaniline on reaction with the peroxide produced by singlet oxygen and histidine as a measure of singlet oxygen production (37). Inhibitors. As mentioned above, many compounds such as carotene, DABCO, and azide, are effective quenchers for singlet oxygen. These compounds, and others which react with singlet oxygen, are frequently used to inhibit reactions in which singlet oxygen is thought to be a reactive intermediate. Care must be taken in interpretation of the results, however, because of their lack of specificity inhibitors that partly avoid calculating the amount of singlet oxygen expected to be inhibited from known rate constants and comparing it with that observed ( 2 4 ) . The quantitative kinetic technique cannot be used in inhomogeneous solutions, where the local concentration of the inhibitor cannot be calculated. Dj>0 Effect- Singlet oxygen has a longer lifetime in D2O than in H2O (iL. 22). Thus many reactions of singlet oxygen proceed more efficiently in D2O than in H 0 . However, there are two important limitations to this technique. First, singlet oxygen reactions in the two solvents will differ in efficiency only if solvent quenching of singlet oxygen limits its lifetime; if substrate or quencher is already removing all the 0 2 , there will be no effect of deuteration on the lifetime. Secondly, it has been shown that 0 " also has a longer lifetime in D 0 than in H 0 (2£L), and reactions of superoxide ion would therefore also be expected to be more efficient in the deuterated solvent. The effect of solvent deuteration on other possible reactive species has not been shown. Thus, this effect cannot be used to distinguish between reactions of 0 a n d 0 " . 2

1

2

2

1

2

2

2

" C l e a n " S o u r c e s of Singlet O x y g e n , One useful technique for studying

suspected singlet oxygen reactions is to generate singlet oxygen under carefully defined conditions free of any other reactive species, and compare its effects with those of the susjpect system. Photochemical systems (using unreactive sensitizers, at high 6 pressure, and with low concentrations of substrates that are unreactive in the Type I reaction) can often be used. Another technique is to use a reverse Diels-Alder reaction, using a naphthalene endoperoxide (40); this technique can be used under very mild conditions (37 °C, neutral), and no side reactions have yet been reported. Most other known chemical sources of singlet oxygen {e. g., hypochlorite/H 02, phosphite ozonides (4JJ) involve very strong oxidants which can react with singlet oxygen substrates. 2

2

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

32

LIGHT-ACTIVATED PESTICIDES

L u m i n e s c e n c e . D i m o l (visible) l u m i n e s c e n c e m a y b e s p e c i f i c for singlet o x y g e n , if the w a v e l e n g t h is carefully m e a s u r e d (42), but c a n not b e easily u s e d to determine the a m o u n t of singlet o x y g e n present, s i n c e it d e p e n d s on a s e c o n d - o r d e r r e a c t i o n b e t w e e n two s i n g l e t o x y g e n m o l e c u l e s . It is e s s e n t i a l that the w a v e l e n g t h of the e m i s s i o n b e carefully d e t e r m i n e d ; in m a n y c a s e s , the s o u r c e s o m e t h i n g other than single D i m o l e m i s s i o n is a l s o difficult to interpret b e c a u s e the extreme sensitivity of photomultipliers a l l o w s the m e a s u r e m e n t of tiny a m o u n t s of light that m a y h a v e little r e l a t i o n s h i p to the m a j o r c h e m i c a l p r o c e s s e s g o i n g o n . T h e infrared l u m i n e s c e n c e of singlet o x y g e n c a n be quantitatively related to the a m o u n t of s i n g l e t o x y g e n p r o d u c e d , a n d c a n l e n d c o n f i d e n c e to its identification if the w a v e l e n g t h is carefully e s t a b l i s h e d (43. 44). A short p u l s e of l a s e r light c a n be u s e d to excite singlet o x y g e n s e n s i t i z e r s , a n d the resulting intensity a n d d e c a y rate of the 1.27^im l u m i n e s c e n c e of singlet o x y g e n c a n b e d e t e c t e d by a g e r m a n i u m p h o t o d i o d e with a lown o i s e a m p l i f i e r a n d a digitizer with s i g n a l a v e r a g i n g ; a s c h e m a t i c of the a p p a r a t u s is s h o w n in F i g . 3 (9. 4 5 . 4 6 ) . T h e a m o u n t of singlet o x y g e n p r o d u c e d a n d its lifetime c a n b e m e a s u r e d very e a s i l y this w a y . T h i s t e c h n i q u e p r o v i d e s a definitive a n d quantitative m e t h o d of c h a r a c t e r i z i n g s i n g l e t o x y g e n p r o d u c e d in p h o t o c h e m i c a l s y s t e m s . F u r t h e r m o r e , by m e a s u r i n g the c h a n g e of lifetime of 0 w h e n a reagent is a d d e d , the rate of its reaction with 0 2 c a n be s i m p l y a n d rapidly d e t e r m i n e d . 1

2

1

T h e y i e l d of singlet o x y g e n p h o t o s e n s i t i z e d by p h o t o d y n a m i c s e n s i t i z e r s c a n b e m e a s u r e d u s i n g this a p p a r a t u s . T h e intensity of the 0 2 l u m i n e s c e n c e is c o m p a r e d with that of a s e n s i t i z e r of k n o w n singlet o x y g e n yield u n d e r c o n d i t i o n s w h e r e the two s e n s i t i z e r s h a v e e q u a l o p t i c a l d e n s i t y . T h e s e v a l u e s are c h e c k e d by m e a s u r i n g the amount of a w e l l - c h a r a c t e r i z e d singlet o x y g e n s u b s t r a t e p h o t o l y z e d in a g i v e n time. W i t h correction for the inefficiency of singlet o x y g e n t r a p p i n g (which c a n be c a l c u l a t e d f r o m the k n o w n rate of reaction of the substrate a n d the d e c a y rate of singlet o x y g e n in the s o l v e n t ) , the a m o u n t of singlet o x y g e n p r o d u c e d in a g i v e n time c a n be c a l c u l a t e d . T h i s v a l u e c a n c o n v e r t e d to a q u a n t u m y i e l d by m e a s u r i n g t h e n u m b e r of q u a n t a a b s o r b e d f r o m t h e l a m p in a g i v e n t i m e by c o n v e n t i o n a l actinometry. T h e infrared l u m i n e s c e n c e determination m e a s u r e s the l o s s of singlet o x y g e n a n d nothing e l s e , s o that it is p o s s i b l e to m e a s u r e a b s o l u t e rates of s i n g l e t o x y g e n r e a c t i o n s with b i o l o g i c a l a c c e p t o r s with c o n f i d e n c e a n d 1

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Mechanisms of Photodynamic Action

2. FOOTE

33

simplicity. T h e sensitivity a n d time r e s p o n s e must be o p t i m i z e d for the timer e s o l v e d s y s t e m to be u s a b l e in a q u e o u s m e d i a , w h e r e the lifetime of singlet o x y g e n is m u c h shorter than in o r g a n i c s o l v e n t s . Transient Absorption Spectroscopy. Transient absorption s p e c t r o s c o p y is useful for m e a s u r i n g m e a s u r i n g the absorption of both radical ions a n d triplet m o l e c u l e s o n a n a n o s e c o n d time s c a l e (47. 48). T h e s e n s i t i z e r is e x c i t e d by a short p u l s e of light, usually from a laser, a n d the a b s o r b a n c e of the transient s p e c i e s m e a s u r e d by a l a m p / p h o t o d e t e c t o r s y s t e m , s h o w n s c h e m a t i c a l l y in F i g . 4. T h i s a p p a r a t u s is u s e f u l for o b s e r v i n g transient intermediates from p h o t o d y n a m i c s e n s i t i z e r s or a c c e p t o r s u n d e r g o i n g T y p e I r e a c t i o n if either the r e d u c e d s e n s i t i z e r o r the o x i d i z e d a c c e p t o r h a s a m e a s u r a b l e a b s o r b a n c e , a s most do. C o n d u c t i v i t y . T i m e - r e s o l v e d c o n d u c t i v i t y m e a s u r e m e n t s h a v e not previously been u s e d muc study of electron-transfer T y p is w i d e l y u s e d in p u l s e r a d i o l y s i s ( 4 9 ) . F o r p h o t o c h e m i c a l w o r k , t h e s e n s i t i z e r is e x c i t e d by a p u l s e d light s o u r c e , a n d the c h a n g e in conductivity m e a s u r e d a s a f u n c t i o n of t i m e . T h i s a p p a r a t u s c a n b e u s e d o n a m i c r o s e c o n d o r n a n o s e c o n d t i m e s c a l e by s l i g h t m o d i f i c a t i o n s . T h e sensitivity for detection of i o n s is excellent, in fact better than that of optical techniques. Examples T w o e x a m p l e s of m e c h a n i s t i c s t u d i e s o n p h o t o d y n a m i c p e s t i c i d e s that h a v e b e e n s t u d i e d in u n u s u a l detail will be p r e s e n t e d to illustrate the u s e s of s o m e of the t e c h n i q u e s d e s c r i b e d in this article. C e r c o s p o r i n . T h e f u n g a l p i g m e n t c e r c o s p o r i n , the structure of w h i c h is s h o w n b e l o w , a c t s p h o t o d y n a m i c a l l y o n plant t i s s u e s , c a u s i n g electrolyte l e a k a g e a n d o t h e r d a m a g e ; t h e s e effects p r o b a b l y a i d t h e attack of the f u n g u s o n the plant ( 5 0 . 5 1 ) . T h i s pigment c a u s e s lipid p e r o x i d a t i o n in the p r e s e n c e of light a n d o x y g e n , a n d the a c t i o n s p e c t r u m for the d a m a g i n g effects is the s a m e a s the absorption s p e c t r u m of c e r c o s p o r i n (52). OH

O OCH

3

CH CHOHCH

3

CH CHOHCH

3

2

2

OCH3 OH

O

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

LIGHT-ACTIVATED PESTICIDES

(

N Computer

\

Digitizer Signal Avg.

I-Amplifiers

\/ Filters

| — Photodiode

Laser

-d

—Cell

Fig. 3. Singlet Oxygen Detection

Computer v

^

i^Amplifiers Digitizer Signal Avg.

Filter

]— Photodiode or PM Tube -Monochromator

Laser

-Cell

Fig. 4. Transient Absorption Spectroscopy

f

^ Computer

V

J

Amplifier

Digitizer Signal Avg.

IBIIIDDDl 000010000

-Blank L-|

Bridge

, Light Source

1

|

i —i— Cell

Fig. 5. Conductivity Apparatus

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

2.

FOOTE

35

Mechanisms of Photodynamic Action

T h e p r o d u c t s of p h o t o o x i d a t i o n of o l e i c a n d l i n o l e b a c i d s a n d of c h o l e s t e r o l s e n s i t i z e d by this pigment w e r e identical to t t o s e with singlet o x i d a t i o n (53. 54). T h e oxidation is inhibited by carotenaids a n d D A B C O (52). D a m a g e is a l s o inhibited by v a r i o u s p h e n o l i c antioxidants (53), but this m a y b e c a u s e d by inhibition of r a d i c a l c h a i n a u t o x i d a t b n of the lipids by b r e a k d o w n of the initial p e r o x i d e s . T h e c h a r a c t e r i s t i c 1.27 n.m singlet o x y g e n e m i s s i o r is readily o b s e r v e d w h e n c e r c o s p o r i n s o l u t i o n s in C D are irradiated (59© q u a n t u m yield of singlet o x y g e n is 0 . 8 1 , a s d e t e r m i n e d by comparison with m e s o - p o r p h y r i n IX d i m e t h y l ester. T h i s v a l u e w a s c o n f i r m e d by 2-rrethyl-2-pentene photo oxidation. T h

6

6

T e r t h i e n y L a - T e r t h i e n y l ( a - T ) is a m e m b e r of a c l a s s of p h o t o d y n a m i c s e n s i t i z e r s , t h e p o l y a c e t y l e n e s , w h i c h a r e present in a n u m b e r of plant s p e c i e s (5£). T h e plants a g a i n s t insect attack; afte killed p h o t o d y n a m i c a l l y . ( H o w e v e r , the nemacocidal activity o b s e r v e d with this c o m p o u n d is difficult to e x p l a i n o n n e b a s i s of a p h o t o d y n a m i c m e c h a n i s m , b e c a u s e no a p p r e c i a b l e light would be e x p e c t e d to penetrate the soil to the depth of the n e m a t o d e s . ) a - T h a s b e e n s h o w n to kill a wide variety of c e l l s , a n d , a l t h o u g h there h a s b e e n s o m e d i s a g r e e m e n t o n this score, t h e r e is a r e q u i r e m e n t for o x y g e n (57. 58). T h e action s p e c t r u m fcr the d a m a g i n g effects is the s a m e a s the absorption s p e c t r u m of a - T .

T h e m e c h a n i s m of a c t i o n of ;his c o m p o u n d h a s b e e n r e v i e w e d (57). T h e r e is c o n s i d e r a b l e c h e m i c a l evidence that singlet o x y g e n is p r o d u c e d by a - T o n irradiation with n e a r - U V Ight. Inhibition of the effects by inhibitors of o t h e r r e a c t i v e o x y g e n s p e c i e s is not o b s e r v e d , but a v a r i e t y of s i n g l e t o x y g e n q u e n c h e r s p r o t e c t a g a i n s t d e a c t i v a t i o n of e n z y m e s by t h i s c o m p o u n d , a n d t h e r e is a p o s i t i v e D 0 effect o n t h e d e a c t i v a t i o n of 2

e n z y m e s . T h e d i o x e t a n e , a typical singlet o x y g e n product, c a n be f o r m e d by a - T - s e n s i t i z e d photooxidation of a d a m a n t y l i d e n e a d a m a n t a n e . D i f f e r e n c e s b e t w e e n the b i o l o g i c a l activities of a - T a n d the singlet o x y g e n s e n s i t i z e r m e t h y l e n e blue h a v e been o b s e r v e d , but they m a y be d u e to d i f f e r e n c e s in localization b e t w e e n the lipophilic a - T a n d the polar m e t h y l e n e blue. T h e f l u o r e s c e n c e yield of a - T in v a r i o u s s o l v e n t s is l e s s t h a n 0 . 1 , a n d the triplet yield is sub&'antial, o n the order of 0.2. T h e singlet o x y g e n yield in e t h a n o l w a s reported to be b e t w e e n 0.15 a n d 0.2 (58). Singlet o x y g e n production by a - T is o b s e r v e d by 1.27 [im e m i s s i o n (R. K a n n e r a n d C . S . F o o t e , in p r e p a r a t i o n ) . T h e q u a n t u m y i e l d of s i n g l e t o x y g e n production is high in b e n z e n e , a s e s t a b l i s h e d by c o m p a r i s o n of the l u m i n e s c e n c e yield with that of s e v e r a l s e n s i t i z e r s with k n o w n q u a n t u m

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

36

LIGHT-ACTIVATED PESTICIDES

y i e l d s of s i n g l e t o x y g e n production. W e a r e currently attempting to determine this q u a n t u m yi*ld m o r e p r e c i s e l y , but p r e s e n t results s u g g e s t t h e y i e l d is around 0.8. It s not certain w h y o u r results differ from t h o s e of K a g a n , S a n t u s et a l ; the s o l v e n t is different, a n d t h e s e a u t h o r s u s e d a s o m e w h a t i n d i r e c t m e t h o d of d e t e r m i n i n g t h e q u a n t u m y i e l d of s i n g l e t o x y g e n formation, the d i s a p p e a r a n c e of d i p h e n y l i s o b e n z o f u r a n . A very recent p a p e r h a s r e p o r t e d t h e kinglet lifetime of a - T t o b e v e r y s h o r t , a n d h a s c h a r a c t e r i z e d t h e p t o t o p h y s i c a l p r o p e r t i e s of both t h e singlet a n d triplet

(52). Summary P r o d u c t i o n of singlet oxygen from t h e s e both c e r c o s p o r i n a n d a - T h a s b e e n u n e q u i v o c a l l y demonstrated. S i n c e in both c a s e s , t h e p h y s i o l o g i c a l effects of t h e p h o t o d y n a m i c actio inhibited by singlet o x y g e c o n d i t i o n s for t h e i n t e r m e d k c y of s i n g l e t o x y g e n in t h e a c t i o n of t h e s e c o m p o u n d s a p p e a r to b e present. Acknowledgments T h e original work reported in this p a p e r w a s s u p p o r t e d by grants from t h e NIH a n d N S F .

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Straight, R. C.; Spikes, J. D. In Singlet O ; Frimer, A. A. Ed.; C R C : Boca Raton, Fla. 1985; Vol. IV, 85-144. Elstner, E. F. Ann. Rev. Plant Physiol. 1982, 33, 73-96. Gollnick, K. Advan. Photochem. 1968, 6, 1-122. Foote, C. S. Free Radicals in Biology 1976, 2, 85-133. Mattes, S. L . ; Farid, S. Science 1984, 226, 917-21. Mattes, S. L . ; Farid, S. In Organic Photochemistry: Padwa, A. Ed.; Marcel Dekker: New York, 1983; 233-326. Foote, C. S. Tetrahedron 1985. 41, 2221-7. Wilkinson, F . ; Brummer, J. G. J. Phys Chem. Ref. Dat. 1981, 10, 809-1000. Monroe, B. In Singlet O ; Frimer, A. A. E d . ; CRC: Boca Raton, Fla. 1985; Vol. I, pp 177-224. Gollnick, K . ; Kuhn, H. J. In Singlet Oxygen: Wasserman, H. H . ; Murray, R. W . , Eds.; Academic: New York, 1979; pp 287-429. Frimer, A. A. In The Chemistry of Peroxides. Patai. S., E d . ; J. Wiley and Sons:, New York, 1983; Chapter 7. Bartlett, P. D . ; Landis, M. In Singlet Oxygen: Wasserman, H. H . ; Murray, R. W . , Eds.; Academic: New York , 1979; pp 244-86. Ando, W . ; Takata, T. In Singlet O ; Frimer, A. A. E d . ; CRC: Boca Raton, Fla. 1985; Vol. III, pp 1-118. 2

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14. Saito, I.; Matsuura, T. In Singlet Oxvaen; Wasserman, H. H . ; Murray, R. W . , Eds.; Academic: New York, 1979; pp 511-74. 15. Foote, C. S. In Singlet Oxygen: Wasserman, H. H.; Murray, R. W . , Eds.; Academic: New York, 1979; pp 139-73. 16. Foote, C. S.; Ching, T.-Y.; Geller, G. G. Photochem. Photobiol. 1974, 20, 511-4. 17. Stevens, B . ; Small, R. D . ; Perez, S. R. Photochem. Photobiol. 1974, 20, 515-8. 18. Fahrenholtz, S. R.; Doleiden, F. H . ; Trozzolo, A. M . ; Lamola, A. A. Photochem. Photobiol. 1974, 20, 505-9. 19. Kasha, M . ; Brabham, D. T. In Singlet Oxygen: Wasserman, H. H.; Murray, R. W . , Eds.; Academic: New York, 1979; pp 1-34. 20. Valenzeno, D. P. Photochem. Photobiol. 1983, 37S, 105-. 21. Bellin, J. S.; Grossman, L. I. Photochem. Photobiol. 1965, 4, 45-53. 22. Steichen, D. S.; Foote 1855-7. 23. Gollnick, K . ; Schnatterer, A. Photochem. Photobiol. 1986, 43, 365-78. 24. Foote, C. S. In Biochemical and Clinical Aspects of Oxygen: Caughey, W. S., E d . ; Academic: New York, 1979; pp 603-26. 25. Krinsky, N. I. In Oxygen Radicals in Chemistry and Biology: Bors, W . , Saran, M . ; Tait, D . , Eds., DeGruyter: Berlin, 1984; 453-64. 26. Krinsky, N. I. In Singlet Oxygen: Wasserman, H. H.; Murray, R. W . , Eds.; Academic: New York, 1979; pp 597-642. 27. Lamola, A. A . ; Yamane, T . ; Trozzolo, A. M. Science 1973, 179, 1131-3. 28. Doleiden, F. H . ; Fahrenholtz, S. R.; Lamola, A. A . ; Trozzolo, A. M. Photochem. Photobiol. 1974, 20, 519-21. 29. Singh, A. Can. J. Phvsiol. Pharm. 1982, 60, 1330-45. 30. Kulig, M. J.; Smith, L. L. J. Org. Chem. 1973, 22, 3639-42. 31. Foote, C. S.; Shook, F. C.; Abakerli, R. A. Meth. Enzymol. 1984, 105, 36-47. 32. Schaap, A. P.; Thayer, A.L.;Faler, G. R.; Goda, K.; Kimura, T. J. Am. Chem. Soc. 1974, 96, 4025-6. 33. Lindig, B. A.; Rodgers, M. A. J.; Schaap, A. P. J. Am. Chem. Soc. 1980, 102, 5590-3. 34. Thomas, M . ; Pryor, W. Lipids 1980, 15, 544-8. 35. Cadet, J.; Decarroz, C . ; Wang, S. Y . ; Midden, W. R. Isr. J. Chem. 1983, 23, 420-9. 36. Lion, Y . ; Delmelle, M . ; Van De Vorst, A. Nature 1976, 263, 442-3. 37. Kralic, I.; El Mohsni, S. Photochem. Photobiol. 1978, 28, 577-81. 38. Kearns, D. R. In Singlet Oxygen: Wasserman, H. H . ; Murray, R. W., Eds.; Academic: New York, 1979; pp 115-38. 39. Bielski, B. H. J.; Saito, E. J. Phys. Chem. 1971, 75, 2263-6. 40. Saito, I.; Matsuura, T . ; Inoue, K. J. Am. Chem. Soc. 1983, 105, 3200-6. 41. Murray, R. W. In Singlet Oxygen: Wasserman, H. H.; Murray, R. W . , Eds.; Academic: New York, 1979; pp 59-114.

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42. Deneke, C. F.; Krinsky, N. I. Photochem. Photobiol. 1977, 25, 299304. 43. Kanofsky, J. Biochem. Biophvs. Res. Comm. 1986. 134, 777-82. 44. Keene, J. P.; Kessel, D.; Land, E. J.; Redmond, R. W.; Truscott, T. G. Photochem. Photobiol. 1986, 43, 117-20. 45. Ogilby, P. R.; Foote, C. S. J. Am. Chem. Soc. 1983. 105, 3423-30. 46. Hurst, J. R.; Schuster, G. B. J. Am. Chem. Soc. 1983. 105. 5756-60. 47. Weir, D . ; Scaiano, J. C.; Arnason, J. T.; Evans, C. Photochem. Photobiol. 1985, 42, 223-30. 48. Malba, V . ; Jones, G. E. II, Poliakoff, E. D. Photochem. Photobiol. 1985, 42, 451-5. 49. Asmus, K. D. Meth. Enzymol. 1984. 105. 167-78. 50. Cavallini, L . ; Bindole, A . ; Macri, F . ; Vianello, A. Chem. Biol. Interact. 1979, 22, 139-46 51. Daub, M. E. Plant Physiol 52. Daub, M. E. Phytopathology 1982, 72, 370-4. 53. Youngman, R. J.; Schieberle, P . ; Schnabl, H . ; Grosch, W . ; Elstner, E. F. Photobiochem. Photobiophys. 1983, 6, 109-19. 54. Daub, M . ; Hangartner, R. P. Plant Physiol. 1983, 72, 855-7. 55. Dobrowolski, D. C.: Foote, C. S. Angew. Chem. 1983, 95, 729-30. 56. Arnason, T . ; Towers, G. H. N . ; Philogene, B. J. R.; Lambert, J. D. H. Am. Chem. Soc. Symposium Ser. 1983, 208. 139-51. 57. Cooper, G. K.; Nitsche, C. I. Bioorg. Chem. 1985, 13, 362-74. 58. Reyftmann, J. P.; Kagan, J.; Santus, R.; Morliere, P. Photochem. Photobiol. 1985. 41, 1-7. 59. Evans, C.; Weir, D . ; Scaiano, J. C.; Mac Eachern, A.; Arnason, J. T.; Morand, P . ; Hollebone, B . ; Leitch, L.C.; Philogene, B. J. R. Photochem. Photobiol. 1986, 44, 441-51. RECEIVED March 10, 1987

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 3

Photomodification and Singlet Oxygen Generation in Membranes Dennis Paul Valenzeno Department of Physiology and K. U. Kidney and Urology Research Center, University of Kansas Medical Center, Kansas City, KS 66103

Photomodification is critical for cell k i l l i n g , is governed by the properties of membrane associated sensitizer. The heterogeneous structure of biological membranes can be an important factor in photosensitization reactions. Sensitizers and protective agents may associate preferentially with the hydrophobic membrane core, may accumulate at the aqueous interface or may bind to membrane proteins. Such localization effects can alter photomodification rates. Although singlet oxygen can diffuse across membrane interfaces in high yield in some cases, membrane associated sensitizer mediates most membrane photomodifications. The membrane environment can influence singlet oxygen generation. Model studies have shown that singlet oxygen quantum yields increase with decreasing solvent polarity. In liposomes or micelles both quantum yields and lifetimes are increased. Aggregation states of sensitizers are changed in the membrane environment leading to alteration of singlet oxygen production. Finally increases in temperature can increase singlet oxygen production due to effects on membrane fluidity.

The goal o f t h i s chapter i s t o d e s c r i b e the c h a r a c t e r i s t i c features of s i n g l e t oxygen g e n e r a t i o n i n membranes as they a r e c u r r e n t l y understood. Membrane p h o t o m o d i f i c a t i o n has been s i n g l e d o u t f o r s p e c i a l c o n s i d e r a t i o n f o r two major reasons. F i r s t recent years have seen an e x p l o s i o n o f i n t e r e s t i n membrane phenomena as the s c i e n t i f i c community has become aware that c e l l u l a r membranes a r e much more than mere gossamer bags t h a t h o l d the i n s i d e i n and the o u t s i d e o u t . Second i n t h e i n s t a n c e s where c e l l u l a r , t i s s u e and organism p h o t o m o d i f i c a t i o n has been examined i n d e t a i l c e l l membranes have r e p e a t e d l y been i d e n t i f i e d as c r i t i c a l t a r g e t s o f m o d i f i c a t i o n ( 1 - 7 ) .

0097-6156/87/0339-0039$06.00/0 © 1987 American Chemical Society

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Membrane Environment:

B i o l o g i c a l membranes. Not o n l y a r e membranes c r i t i c a l cellular components and c r i t i c a l t a r g e t s f o r p h o t o m o d i f i c a t i o n , they a l s o present a unique environment f o r p h o t o s e n s i t i z e r s which generate s i n g l e t oxygen. I n f a c t i f t h i s were not the case we would not need to c o n s i d e r membrane s e n s i t i z a t i o n as a separate topic. The c h a r a c t e r i s t i c s of s i n g l e t oxygen g e n e r a t i o n by s e n s i t i z e r s i n aqueous s o l u t i o n would a p p l y . As we s h a l l see t h i s i s not the case. Membranes present an environment that d i f f e r s from the s u r r o u n d i n g medium not o n l y i n p o l a r i t y , water content and d i e l e c t r i c c o n s t a n t , but they a r e heterogenous s t r u c t u r e s which present a v a r i e t y of domains w i t h which s e n s i t i z e r s can a s s o c i a t e and from which they can act. Current concepts of the s t r u c t u r e of c e l l membranes a r e based on the f l u i d mosaic model of S i n g e r and N i c o l s o n ( 8 ) . In this view, F i g u r e 1A, the membrane b i l a y e r . Their hydrophobic the center of the b i l a y e r e x p o s i n g t h e i r p o l a r head groups t o the aqueous environment a t e i t h e r s u r f a c e . T h i s arrangement is s t a b i l i z e d by the h y d r o p h o b i c f o r c e s between the p h o s p h o l i p i d s and does not i n v o l v e c o v a l e n t bonding. The m a j o r i t y of the p h o s p h o l i p i d s are thus f r e e to d i f f u s e w i t h i n the plane of the membrane, but move with difficulty from one s u r f a c e of the b i l a y e r to the o t h e r . Membrane p r o t e i n s a r e i n s e r t e d i n t o the l i p i d b i l a y e r , e i t h e r p a r t way or e n t i r e l y spanning the b i l a y e r (so c a l l e d i n t e g r a l or i n t r i n s i c proteins). The p o r t i o n s of these p r o t e i n s which a r e i n c o n t a c t w i t h the h y d r o p h o b i c i n t e r i o r of the b i l a y e r a r e composed of a h i g h p r o p o r t i o n of hydrophobic amino a c i d s , w h i l e the p o r t i o n s exposed at the aqueous i n t e r f a c e have a h i g h p r o p o r t i o n of h y d r o p h i l i c amino acids. Thus the p r o t e i n s a r e a l s o s t a b i l i z e d i n p o s i t i o n by h y d r o p h o b i c f o r c e s and have the same a b i l i t y to d i f f u s e i n the p l a n e of the b i l a y e r but not a c r o s s it. Both the p r o t e i n s and p h o s p h o l i p i d s can have c a r b o h y d r a t e groups a t t a c h e d t o them, b u t such groups have been found o n l y at the o u t s i d e s u r f a c e of the c e l l . Most animal c e l l membranes have a v a r i a b l e content of c h o l e s t e r o l i n t e r s p e r s e d w i t h the p h o s p h o l i p i d s . The p r o p o r t i o n of c h o l e s t e r o l i s e s p e c i a l l y h i g h i n the membrane of the r e d b l o o d c e l l , which i s the membrane s t u d i e d most e x t e n s i v e l y . The most s i g n i f i c a n t m o d i f i c a t i o n of these ideas that has occurred i n recent y e a r s has been the d i s c o v e r y that i n many instances i n t e g r a l membrane p r o t e i n s a r e r e s t r i c t e d i n t h e i r m o t i o n by an i n t r a c e l l u l a r s k e l e t o n of p e r i p h e r a l ( o r e x t r i n s i c ) membrane p r o t e i n s that serve to anchor some of the i n t r i n s i c proteins i n l o o s e l y f i x e d p o s i t i o n s . I n the red c e l l the c y t o s k e l e t a l network of p e r i p h e r a l membrane p r o t e i n s l i e s j u s t below the membrane surface and anchors i n t e g r a l p r o t e i n s , which span the b i l a y e r , at p e r i o d i c p o i n t s v i a a p r o t e i n component known as a n k y r i n ( 9 , 1 0 ) . The r e s u l t of the membrane s t r u c t u r e j u s t d e s c r i b e d i s t h a t the i n t e r i o r of the membrane has the c h a r a c t e r i s t i c s of the i n t e r i o r of a lipid bilayer. The d i e l e c t r i c constant ( p o l a r i t y ) i s very low (2-3) in this region. L i p o p h i l i c s o l u t e s can be expected t o p a r t i t i o n readily i n t o t h i s domain. Water i s present i n g r e a t l y reduced c o n c e n t r a t i o n w i t h some i n v e s t i g a t o r s c l a i m i n g t h a t the b i l a y e r i s

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d e v o i d of w a t e r . Water p e r m e a b i l i t y of most membranes i s , however, quite high. The o t h e r s i g n i f i c a n t f e a t u r e of the membrane environment which deserves comment i s the i n t e r f a c i a l r e g i o n . This i s a region e x t e n d i n g r o u g h l y from the g l y c e r o l backbone of the p h o s p h o l i p i d s away from the membrane to the end of the a t t a c h e d c a r b o h y d r a t e m o i e t i e s . T h i s r e g i o n i s of i n t e r m e d i a t e p o l a r i t y between the b i l a y e r i n t e r i o r and the aqueous environment ( d i e l e c t r i c c o n s t a n t of about 10). Due to the p o l a r n a t u r e of the charged groups at the membrane i n t e r f a c e , the s u r f a c e of most b i o l o g i c a l membranes has a net n e g a t i v e charge. T h i s s u r f a c e charge can modify the d i s t r i b u t i o n of charged s o l u t e s near i t . I n p a r t i c u l a r the c o n c e n t r a t i o n of c a t i o n s i s h i g h e r and the c o n c e n t r a t i o n of a n i o n s i s lower w i t h i n a few angstroms of the s u r f a c e than i n the b u l k s o l u t i o n a d j a c e n t to the membrane. I n t r a n s p o r t s t u d i e s i t has even been p o s s i b l e to d i s c e r n the e f f e c t s of l o c a l s u r f a c e charge, i . e . charged groups l o c a t e d near the opening of a p r o t e i n a c e o u Water p r e s e n t near th charged groups i n t o a s t r u c t u r e more l i k e i c e than l i q u i d w a t e r . Membrane model systems. Model systems have been v e r y v a l u a b l e as guides to u n d e r s t a n d i n g the c h a r a c t e r i s t i c s of s i n g l e t oxygen i n membrane systems. However, the r e s u l t s must a l s o be v e r i f i e d i n b i o l o g i c a l membranes s i n c e the assembly of p h o s p h o l i p i d s and p r o t e i n s of a c e l l membrane i s s i g n i f i c a n t l y more complex than most model systems. Still, in many i n s t a n c e s models p r o v i d e the o n l y information currently available. The model systems most o f t e n employed are m i c e l l e s or l i p o s o m e s , ( F i g u r e I B ) . The former are aqueous d i s p e r s i o n s of amphipathic molecules. These m o l e c u l e s which have a h y d r o p h o b i c and h y d r o p h i l i c p o r t i o n spontaneously form aggregates i n aqueous s o l u t i o n such that the i n t e r i o r of the a g g r e g a t e , or m i c e l l e , c o n t a i n s the h y d r o p h o b i c p o r t i o n s and thus mimics the membrane i n t e r i o r . The a r e a of c o n t a c t w i t h water mimics the membrane i n t e r f a c i a l r e g i o n and can be charged of e i t h e r s i g n , or uncharged depending on the s t r u c t u r e of the a m p h i p a t h i c m o l e c u l e used. Liposomes are membranous s t r u c t u r e s which resemble soap b u b b l e s . Many a m p h i p a t h i c m o l e c u l e s w i l l s p o n t a n e o u s l y form such s t r u c t u r e s when a g i t a t e d w i t h an aqueous phase. They can be e i t h e r u n i l a m e l l a r , t h a t i s composed of a s i n g l e b i l a y e r w i t h an e n c l o s e d aqueous phase, or m u l t i - l a m e l l a r , i n which t h e r e are m u l t i p l e b i l a y e r s e n c l o s i n g the aqueous phase. The incorporated aqueous phase can have a d i f f e r e n t c o m p o s i t i o n from the s u s p e n s i o n medium. S e n s i t i z e r - Membrane I n t e r a c t i o n s The a b i l i t y of s e n s i t i z e r s to generate s i n g l e t oxygen i n membranes can be i n f l u e n c e d by i n t e r a c t i o n of the s e n s i t i z e r w i t h membrane components. B i n d i n g of s e n s i t i z e r to s u b s t r a t e has been shown to f a v o r Type I r e a c t i o n s i n homogenous s o l u t i o n s . Modification in which the s e n s i t i z e r i s p h y s i c a l l y s e p a r a t e d from the t a r g e t suggests a Type I I r e a c t i o n . The s e n s i t i z e r s to be c o n s i d e r e d here include the h a l o g e n a t e d f l u o r e s c e i n d e r i v a t i v e s ( x a n t h e n e s ) , the p o r p h y r i n s and merocyanine-540. These were s e l e c t e d because they are w i d e l y

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Intrinsic Anion Transport ^Proteins Protein |r

Ankyrin

<£) 0.05-1^ jjm[) Liposome

F i g u r e 1. Membrane S t r u c t u r e . (A) The r e d b l o o d c e l l membrane. Dimensions a r e approximate. (B) Two model membrane systems. P h o s p h o l i p i d s w i t h t h e i r two hydrocarbon c h a i n s a r e d e p i c t e d as c i r c l e s w i t h two wavy l i n e s .

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studied s i n g l e t oxygen s e n s i t i z e r s which a f f e c t c e l l membranes, but i n some i n s t a n c e s e x h i b i t w i d e l y d i f f e r e n t b e h a v i o r . S e n s i t i z e r E x t e r n a l to the Membrane. The simplest sensitizer membrane i n t e r a c t i o n i s the absence of i n t e r a c t i o n . That i s , the s e n s i t i z e r generates s i n g l e t oxygen i n the aqueous medium e x t e r n a l to the membrane which then d i f f u s e s to the membrane and effects a modification. Within i t s 3 to 4 microsecond l i f e t i m e i n aqueous solution (12-14) s i n g l e t oxygen can d i f f u s e about 0.1 m i c r o n s b e f o r e it decays to the ground s t a t e (15,16). Since this distance i s c o n s i d e r a b l y g r e a t e r than the 0.005 m i c r o n t h i c k n e s s of biological membranes, m o d i f i c a t i o n by s i n g l e t oxygen generated e x t e r n a l to the membrane i s a d i s t i n c t p o s s i b i l i t y . Experimental v e r i f i c a t i o n has been found i n the work of Bezman, £t a l . (17) who demonstrated that b a c t e r i a c o u l d be p h o t o i n a c t i v a t e d by s i n g l e t oxygen generated by rose bengal i m m o b i l i z e d on l a r g e p o l y s t y r e n e beads. I n model systems a l s o s i n g l e t oxygen has been shown to p e n e t r a t e i n t o m i c e l l e s from the aqueous suspension mediu by s i n g l e t oxygen generate the suspension medium (19). [See s e c t i o n D.5. for penetration of singlet oxygen through membranes.] However, with very few exceptions, p h o t o s e n s i t i z a t i o n i n b i o l o g i c a l systems o c c u r s with membrane a s s o c i a t i o n of the s e n s i t i z e r , e i t h e r by d i s s o l u t i o n of the s e n s i t i z e r i n the h y d r o p h o b i c membrane i n t e r i o r or by b i n d i n g to some membrane component ( 4 ^ 20-22). Sensit i z e r Bound to the Membrane. In instances where a p h o t o s e n s i t i z e r i s a t t a c h e d to a l i g a n d w i t h known b i n d i n g p r o p e r t i e s the l o c a l i z a t i o n of s e n s i t i z e r w i t h r e s p e c t to the membrane i s f a i r l y well defined. I n an attempt to l o c a l i z e the c r i t i c a l target for photomodification of e r y t h r o c y t e membranes P o o l e r and G i r o t t i (23) used E o s i n Y a t t a c h e d to an i s o t h i o c y a n a t e m o i e t y , a compound which i s known to b i n d s p e c i f i c a l l y to the membrane p r o t e i n r e s p o n s i b l e f o r anion t r a n s p o r t (and p o s s i b l y t r a n s p o r t of o t h e r m a t e r i a l s ) . While the photochemical p r o p e r t i e s and s i n g l e t oxygen g e n e r a t i n g c a p a b i l i t y of E o s i n - i s o t h i o c y a n a t e were not a l t e r e d from f r e e E o s i n Y, i t was 50 to 100 times as e f f e c t i v e as a s e n s i t i z e r f o r p h o t o h e m o l y s i s . Thus the s i t e of g e n e r a t i o n of the s i n g l e t oxygen was crucial. These r e s u l t s have been i n t e r p r e t e d as an i m p l i c a t i o n f o r a c r i t i c a l role of the d i m e r i c red c e l l a n i o n t r a n s p o r t p r o t e i n i n p h o t o h e m o l y s i s (23,24). Since the p r o d u c t i o n of membrane l e s i o n s l e a d i n g to photohemolysis o c c u r s through the combined a c t i o n of two photons and two s e n s i t i z e r m o l e c u l e s (22, 25-27), a d i m e r i c p r o t e i n i s a l i k e l y target. Another example of the e x p l o i t a t i o n of known b i n d i n g p r o p e r t i e s a r i s e s f o r the s e n s i t i z e r merocyanine-540 (M-540). M-540 was used as an o p t i c a l probe of membrane p o t e n t i a l i n the 70 s (2j8) i n e x c i t a b l e cells. I t was subsequently shown t h a t M-540 has an a f f i n i t y for e x c i t a b l e c e l l s and h e m a t o p o i e t i c c e l l s (29, 3 0 ) , but of even more i n t e r e s t was the d e m o n s t r a t i o n t h a t most of t h i s b i n d i n g i s of r e l a t i v e l y low a f f i n i t y whereas b i n d i n g to leukemic c e l l s i s of very high a f f i n i t y (31.). In a manner s i m i l a r to the treatment of malignancies w i t h hematoporphyrin d e r i v a t i v e and l i g h t , M-540 and light i s now b e i n g developed f o r the treatment of leukemia. In aqueous s o l u t i o n M-540 generates s i n g l e t oxygen upon illumination 1

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(32). I t b i n d s to the s u r f a c e of c e l l membranes (33) and p r e l i m i n a r y evidence suggests that s i n g l e t oxygen i s i n v o l v e d in i t s p h o t o m o d i f i c a t i o n of membranes ( V a l e n z e n o , et a l . , u n p u b l i s h e d results). Sensitizer A s s o c i a t e d w i t h the Membrane. Membrane associated sensitizer i s more important f o r most p h o t o s e n s i t i z a t i o n reactions than s e n s i t i z e r d i s s o l v e d i n the b a t h i n g medium. Bagchi and Basu (34) were a b l e to demonstrate that a c r i f l a v i n e m o l e c u l e s remaining o u t s i d e E. c o l i were i n e f f e c t i v e at p h o t o i n a c t i v a t i o n by s i m p l y diluting the external medium just before illumination. P h o t o i n a c t i v a t i o n proceeded at a r a t e governed by the c o n c e n t r a t i o n of sensitizer i n which the b a c t e r i a were i n c u b a t e d , not the c o n c e n t r a t i o n i n which they were subsequently i l l u m i n a t e d . The commonly used h a l o g e n a t e d f l u o r e s c e i n sensitizers, which i n c l u d e both e o s i n Y and rose b e n g a l , a l s o produce p h o t o m o d i f i c a t i o n while associated with membranes. The action spectra for p h o t o m o d i f i c a t i o n of l o b s t e t h i s c l a s s show red s h i f t aqueous s o l u t i o n (315). An a l t e r a t i o n of the environment of the s e n s i t i z e r as would o c c u r upon a s s o c i a t i o n w i t h the membrane can e x p l a i n the red s h i f t . The l o c a l i z a t i o n of the s e n s i t i z e r w i t h i n the membrane has not been w e l l s t u d i e d . Evidence must be gleaned from studies i n a v a r i e t y of f i e l d s . Varnadore, et a l . , (36) have measured p h o t o - v o l t a g e s produced by e r y t h r o s i n B, a h a l o g e n a t e d f l u o r e s c e i n , a c r o s s b i l a y e r membranes. T h e i r r e s u l t s suggest t h a t e r y t h r o s i n B l o c a l i z e s at the plane of the g l y c e r o l r e g i o n of the membrane p h o s p h o l i p i d s . Other h a l o g e n a t e d f l u o r e s c e i n s , a l t h o u g h not s t u d i e d as t h o r o u g h l y , y i e l d e d s i m i l a r p h o t o - v o l t a g e s . This i s c o n s i s t e n t w i t h o x i d a t i o n - r e d u c t i o n r e a c t i o n s mediated by erythrosin B i n b r a i n membranes (3^7). I n m i c e l l e s both rose bengal and e r y t h r o s i n B l o c a l i z e near the i n t e r f a c e (38-39) as do merocyanines (40). U s i n g merocyanine-540 L e l k e s and M i l l e r (33) have shown a l o c a l i z a t i o n near the g l y c e r o l r e g i o n of c e l l membranes. The r e l a t i v e potency of ten d i f f e r e n t h a l o g e n a t e d fluorescein sensitizers f o r membrane p h o t o m o d i f i c a t i o n v a r i e s over a range of 5,000 t o 3 5 , 0 0 0 - f o l d f o r red c e l l s and nerve c e l l s respectively (21 ,22). Yet the photochemical p r o p e r t i e s and r e l a t i v e e f f e c t i v e n e s s for enzyme i n a c t i v a t i o n i n aqueous s o l u t i o n d i f f e r by o n l y a factor of about 20 (41). The d i s c r e p a n c y can a g a i n be a t t r i b u t e d to s e n s i t i z e r l o c a l i z a t i o n and d i f f e r i n g photochemical p r o p e r t i e s i n the membrane environment. I n the case of c e l l membranes the relative e f f e c t i v e n e s s of the h a l o g e n a t e d f l u o r e s c e i n s was accounted f o r by assuming t h a t s e n s i t i z e r p a r t i t i o n s between the aqueous s u s p e n s i o n medium and the membrane, and then absorbs l i g h t and c r e a t e s s i n g l e t oxygen i n t h a t environment. The r e s u l t s of modeling based on these assumptions are c o n s i s t e n t w i t h the observed v a r i a t i o n i n potency, F i g u r e 2. T h i s v a r i a t i o n cannot be accounted f o r i f i t i s assumed t h a t s e n s i t i z e r a c t s from the s u s p e n s i o n medium. These results suggest t h a t s e n s i t i z e r photochemical p r o p e r t i e s and a b i l i t y to generate s i n g l e t oxygen may be d i f f e r e n t i n membranes. We will c o n s i d e r t h i s i n some d e t a i l below. A s s o c i a t i o n of s e n s i t i z e r w i t h membranes, a s s e s s e d as s e n s i t i z e r h y d r o p h o b i c i t y , has been shown to be a c r i t i c a l determinant of sensitizing efficacy f o r p o r p h y r i n s as w e l l as halogenated

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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FL

DCFL DBFL

EB

EY

ERY

PX

PB

ERB

RB

F i g u r e 2. R e l a t i v e e f f e c t i v e n e s s of h a l o g e n a t e d f l u o r e s c e i n s . The bars r e p r e s e n t the measured v a l u e s of r e l a t i v e e f f e c t i v e n e s s f o r p h o t o h e m o l y s i s of the s e n s i t i z e r s l i s t e d . The p o i n t s a r e the p r e d i c t e d e f f e c t i v e n e s s v a l u e s based on a model t h a t assumes t h a t the s e n s i t i z e r a c t s from the membrane environment, not the aqueous susp e n s i o n medium. FL = f l u o r e s c e i n ; PX = t e t r a b r o m o d i c h l o r o f l u o r e s c e i n ; a l l o t h e r a b b r e v i a t i o n s a r e d e f i n e d i n F i g u r e 3. Adapted from Ref. 22.

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f l u o r e s c e i n s ( 2 ^ 42,43). Mesoporphyrin accumulates i n a r e g i o n w i t h a d i e l e c t r i c constant of about 20 i n L1210 c e l l s from which i t i s an a c t i v e s e n s i t i z e r (44). Such a d i e l e c t r i c constant suggests an intramembranous l o c a l i z a t i o n near the i n t e r f a c e as was the case f o r the fluorescein derivatives. Suwa, et a l . (45) demonstrated t h a t hematoporphyrin s e n s i t i z e d the o x i d a t i o n of membrane c h o l e s t e r o l much more e f f i c i e n t l y when i t was i n c o r p o r a t e d i n t o the membrane phase of liposomes than when i t was d i s s o l v e d i n the aqueous s u s p e n s i o n medium. A d d i t i o n a l l y , i t has been shown t h a t the most h y d r o p h o b i c f r a c t i o n s of the tumor s e n s i t i z e r m i x t u r e , known as hematoporphyrin derivative, which p a r t i t i o n best i n t o membranes are the most effective sensitizers (46, 47). Action spectra for both hematoporphyrin and i t s a c t i v e f r a c t i o n l e a d to the s u g g e s t i o n that these sensitizers are bound to membrane proteins during photoexcitation (46). F i n a l l y , we should note t h a t both b i n d i n g and p a r t i t i o n i n g may be important f o r membran p h o t o s e n s i t i z i n g potenc above, i s p r o p o r t i o n a l to t h e i r a b i l i t y to p a r t i t i o n between aqueous medium and membrane-like l i p i d s o l v e n t s . Recent work, however, has shown t h a t the f i n a l d i s t r i b u t i o n of s e n s i t i z e r does not follow a simple p a r t i t i o n i n g i s o t h e r m , but i n v o l v e s b i n d i n g of the s e n s i t i z e r to membrane s i t e s (26, 2 7 ) . S i m i l a r c o n c l u s i o n s based on c o m p l e t e l y d i f f e r e n t e x p e r i m e n t a l e v i d e n c e have been drawn f o r some porphyrins (46). C h a r a c t e r i s t i c s of S i n g l e t Oxygen i n Membranes Biochemistry of S i n g l e t Oxygen M o d i f i c a t i o n of Membranes. Singlet oxygen i s capable of m o d i f y i n g many components of biological membranes. John Spikes w i l l d i s c u s s the b i o c h e m i s t r y of photodynamic a c t i o n i n Chapter 6, but a b r i e f d i s c u s s i o n i s i n order here so t h a t the mechanisms of s i n g l e t d e t e c t i o n i n membranes w i l l be u n d e r s t a n d able. U n s a t u r a t e d bonds i n p h o s p h o l i p i d s are s u s c e p t i b l e to a t t a c k by singlet oxygen l e a d i n g to a v a r i e t y of p e r o x i d i z e d lipids. Malonaldehyde, a product of p o l y u n s a t u r a t e d f a t t y a c i d o x i d a t i o n , i s readily d e t e c t a b l e u s i n g a simple c o l o r i m e t r i c method ( 4 8 ) . Side chains of f i v e amino a c i d s are p h o t o m o d i f i a b l e . These include histidine, t y r o s i n e , tryptophan, c y s t e i n e and methionine. More extensive m o d i f i c a t i o n can produce p r o t e i n c r o s s - l i n k i n g which i s more e a s i l y d e t e c t e d by g e l e l e c t r o p h o r e s i s . F i n a l l y c h o l e s t e r o l can be m o d i f i e d by s i n g l e t oxygen to produce the c h a r a c t e r i s t i c 5 alpha h y d r o p e r o x i d e of c h o l e s t e r o l . Only s i n g l e t oxygen i s known to produce t h i s o x i d a t i o n product of c h o l e s t e r o l . For a more d e t a i l e d d i s c u s s i o n of s i n g l e t oxygen c h e m i s t r y , see Chapter 2 by C h r i s t o p h e r Foote. D e t e r m i n a t i o n of the i n t e r m e d i a c y of s i n g l e t oxygen i n membrane m o d i f i c a t i o n s f o l l o w s the methods a v a i l a b l e i n s o l u t i o n . T y p i c a l l y a quencher such as a z i d e or a r e a c t a n t such as a f u r a n are used to compete w i t h s o l v e n t quenching and r e a c t i o n w i t h membrane-located substrate. A reduction i n m o d i f i c a t i o n suggests a s i n g l e t oxygen mechanism. C o n v e r s e l y , an i n c r e a s e i n m o d i f i c a t i o n when d e u t e r i u m oxide r e p l a c e s water (which quenches s i n g l e t oxygen) i n d i c a t e s a s i n g l e t oxygen r e a c t i o n . [Deuterium o x i d e i s known to increase

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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s i n g l e t oxygen l i f e t i m e s compared to aqueous s o l u t i o n about 15-fold (13, 4 9 ) ] . I n membrane systems, however, t h e r e i s t h e added c o m p l e x i t y o f a c c e s s of the m o d i f y i n g agent t o the membrane. Some q u e n c h e r s / r e a c t a n t s ( b e t a - c a r o t e n e and a l p h a - t o c o p h e r o l ) a r e v e r y lipid s o l u b l e and p a r t i t i o n r e a d i l y i n t o membranes. Others (azide, i m i d a z o l e ) a r e not l i k e l y t o p e n e t r a t e i n t o the hydrocarbon core o f the b i l a y e r . Deuterium o x i d e e f f e c t s may a l s o be s e v e r l y reduced s i n c e the r e a c t i o n s may not be o c c u r r i n g i n an aqueous environment. For these reasons i t i s frequently observed that higher c o n c e n t r a t i o n s of quenchers, r e a c t a n t s and/or d e u t e r i u m o x i d e a r e r e q u i r e d f o r an o b s e r v a b l e e f f e c t i n membrane systems ( 1 5 , 50-52). S i n g l e t Oxygen Quantum Y i e l d s The quantum y i e l d of s i n g l e t oxygen f o r m a t i o n (number of s i n g l e t oxygen m o l e c u l e s generated per absorbed photon) i s f r e q u e n t l y found to be h i g h e r i n membranes than i n aqueous media f o r p o r p h y r i n s , w h i l e c u r r e n t e v i d e n c e suggests the o p p o s i t e for fluorescein s e n s i t i z e r s showed that i n t h e p o r p h y r i n s s i n g l e t oxygen quantum y i e l d s were i n c r e a s e d compared t o the r e s u l t s i n phosphate b u f f e r . They a t t r i b u t e d t h i s e f f e c t t o solubilization and monomerization of their sensitizers (hematoporphyrin and t h e a c t i v e f r a c t i o n o f hematoporphyrin d e r i v a t i v e ) by the l i p o s o m e s . The quantum y i e l d s f o r hematoporphyrin were doubled w h i l e they were i n c r e a s e d about 1 5 - f o l d f o r the a c t i v e f r a c t i o n of hematoporphyrin d e r i v a t i v e . The q u a n t i t a t i v e r e s u l t s of t h i s study must be viewed w i t h c a u t i o n , however, s i n c e i t i s based on the assumption that s i n g l e t oxygen quantum y i e l d s s e n s i t i z e d by rose bengal a r e t h e same i n aqueous s u s p e n s i o n as i n membranes ( s e e b e l o w ) . Such a decrease i n s i n g l e t quantum y i e l d s due t o a g g r e g a t i o n of s e n s i t i z e r has been found i n o t h e r s t u d i e s under a v a r i e t y o f solvent/model membrane c o n d i t i o n s (14, 54, 5 5 ) . I n a study by R e d d i , et a l . , (56) i t was noted that t r i p l e t quantum y i e l d s o f t h e sensitizer i n m i c e l l e s were s i m i l a r to those found i n organic s o l v e n t s f o r both c o p r o p o r p h y r i n and h e m a t o p o r p h y r i n , both b e i n g e l e v a t e d from the v a l u e s i n aqueous media. However, the a b i l i t y of a sensitizer triplet t o generate s i n g l e t oxygen was about t w i c e as l a r g e i n the o r g a n i c s o l v e n t as i n e i t h e r the m i c e l l e s o r aqueous buffer. The reasons f o r t h i s s o l v e n t e f f e c t on s i n g l e t oxygen g e n e r a t i o n a r e n o t c l e a r , but cannot be a t t r i b u t e d to micellar s u r f a c e charge o r o r g a n i c s o l v e n t p o l a r i t y s i n c e the r e s u l t s were i n v a r i a n t when these parameters were changed. Similar observations of solvent-dependent i n c r e a s e s i n s i n g l e t oxygen quantum y i e l d s , i n excess o f t h a t which can be e x p l a i n e d by a g g r e g a t i o n e f f e c t s have been a t t r i b u t e d t o c h e m i c a l e f f e c t s on the p o r p h y r i n s i d e c h a i n s (14). T h i s l a s t study p r o v i d e s an i n t e r e s t i n g d e m o n s t r a t i o n of the importance of the solvent f o r singlet quantum yields. Hematoporphyrin was d i s s o l v e d i n t o t h e aqueous phase o f an o c t a n o l / w a t e r system and was a l l o w e d to d i s t r i b u t e between the two phases. A narrow beam of l i g h t was used t o i l l u m i n a t e a s m a l l a r e a of s o l u t i o n and the 1.27 m i c r o n luminescence produced by s i n g l e t oxygen was m o n i t o r e d . When the e x c i t i n g beam was moved a c r o s s t h e phase boundary from water t o o c t a n o l a marked increase i n luminescence was d e t e c t e d i n d i c a t i n g a h i g h e r s i n g l e t oxygen concentration.

American Chemical Society Library 1155 16th St., N.W. In Light-Activated Pesticides; Washington, D.C. Heitz, 20036J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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PORPHYRINS

1

SOLVENT

0 Quantum Yield 2

PBS SUV/PBS MeOH/D 0 Form/D^l CTAB/Dp SDS/D 0

HP

2

2

HPD 0.9%NaCI PBS MeOH

XANTHENES FL l CI (RB) 4

4

FL Br CI (PB) 4

4

FL l (ERB) 4

H0 MeOH H/) MeOH HjO MeOH 2

FL Br (EY) 4

MeOH FLBr (N0 ) (EB) H 0 MeOH H0 FL l MeOH HjO FL Br MeOH H0 FL Br (DBFL) MeOH H0 FLCI (DCFL) MeOH H0 FL C l MeOH H0 FL MeOH 2

2

2

2

2

2

3

2

2

2

2

4

2

2

0.2 0.4 0.6

F i g u r e 3. S i n g l e t oxygen quantum y i e l d s o f s e l e c t e d p o r p h y r i n s and h a l o g e n a t e d f l u o r e s c e i n s ( x a n t h e n e s ) . S i n g l e t quantum y i e l d s a r e p l o t t e d i n v a r i o u s s o l v e n t systems as a v a i l a b l e i n the l i t e r a t u r e . Adapted from R e f s . 53, 54, 56, and 58.

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With f l u o r e s c e i n s e n s i t i z e r s t r i p l e t quantum y i e l d s , singlet oxygen quantum y i e l d s and s e n s i t i z i n g e f f e c t i v e n e s s a l l i n c r e a s e w i t h the degree of h a l o g e n a t i o n of the s e n s i t i z e r (21, 22, 57, 5 8 ) . The triplet quantum y i e l d i n c r e a s e i s due to the heavy atom e f f e c t of h a l o g e n a t i o n l e a d i n g to an i n c r e a s e i n i n t e r s y s t e m c r o s s i n g from the excited s i n g l e t s t a t e of the s e n s i t i z e r to the e x c i t e d t r i p l e t ( 5 9 ) . Increases i n s i n g l e t oxygen y i e l d and sensitizing effectiveness f o l l o w from i n c r e a s e d t r i p l e t y i e l d . Studies with f l u o r e s c e i n s using organic s o l v e n t s to mimic membranes have g e n e r a l l y shown decreases i n s e n s i t i z e r triplet and s i n g l e t oxygen quantum y i e l d s i n these s o l v e n t s . I n c o n t r a s t to the porphyrins, f l u o r e s c e i n s do not aggregate i n the m i c r o m o l a r to m i l l i m o l a r range ( 5 8 ) . Thus there i s no a g g r e g a t i o n e f f e c t on the yields. Rather s h i f t s i n the e l e c t r o n i c energy l e v e l s w i t h s o l v e n t become important (58). These l e a d to a r e d u c t i o n i n t r i p l e t s e n s i t i z e r quantum y i e l d s and s i n g l e t oxygen quantum y i e l d s i n media l e s s p o l a r than w a t e r y i e l d s are lower i n a e r a t e on their l i t e r a t u r e review, these i n v e s t i g a t o r s assumed that a l l s e n s i t i z e r t r i p l e t s are quenched by g e n e r a t i o n of s i n g l e t oxygen i n a e r a t e d e t h a n o l but that s i n g l e t oxygen i s produced i n o n l y about 70% of the t r i p l e t quenchings i n aqueous s o l u t i o n s . A s i m i l a r p a t t e r n of decreased t r i p l e t quantum y i e l d i n a s o l v e n t of lower polarity, but with concommitant dimerization, has been demonstrated for c h l o r o p h y l l s a and b by Bowers and P o r t e r ( 6 0 ) . S i n g l e t Oxygen L i f e t i m e s . I n a d d i t i o n to the t o t a l y i e l d of s i n g l e t oxygen i t s l i f e t i m e w i l l i n f l u e n c e the degree of r e a c t i o n . The l o n g e r oxygen remains i n an e x c i t e d s t a t e the more l i k e l y i t i s to modify a g i v e n t a r g e t . The membrane environment can a f f e c t singlet oxygen l i f e t i m e s and thus a l t e r the r e a c t i o n p r o b a b i l i t y and mean diffusion d i s t a n c e of the e x c i t e d s t a t e . For example i n a hydrocarbon environment l i k e the i n t e r i o r of a membrane P o o l e r and Valenzeno (J_5) c a l c u l a t e d t h a t the l e n g t h c o n s t a n t ( d i s t a n c e at which the c o n c e n t r a t i o n of s i n g l e t oxygen has f a l l e n to 1/e of i t s c o n c e n t r a t i o n at i t s source) f o r s i n g l e t oxygen d i f f u s i o n was i n c r e a s e d about 2 - 1 / 2 - f o l d over that i n aqueous s o l u t i o n . T h i s means t h a t 37% of the s i n g l e t oxygen generated w i l l be a b l e to d i f f u s e about 0.25 micrometers b e f o r e l o s i n g i t s energy of e x c i t a t i o n . Since t h e r e are no e x p e r i m e n t a l l y determined v a l u e s f o r s i n g l e t oxygen l i f e t i m e i n b i o l o g i c a l membranes the r e s u l t s of model studies provide the only i n f o r m a t i o n , F i g u r e 4. I n the o r g a n i c solvent, formamide, s i n g l e t oxygen l i f e t i m e i s reported to be increased compared to the 3 to 4 microsecond l i f e t i m e i n aqueous s o l v e n t s ( 6 1 ) . I n m i c e l l e s s i n g l e t l i f e t i m e s are i n c r e a s e d to v a r y i n g degrees. In both a n i o n i c and c a t i o n i c m i c e l l e s s i n g l e t l i f e t i m e s have been shown t o be i n c r e a s e d to 20-25 microseconds ( 6 2 ) . These v a l u e s were the same as those found i n the c o r r e s p o n d i n g pure hydrocarbon l a c k i n g the charged head group. Thus i n t h i s case charged groups at the i n t e r f a c e were u n i m p o r t a n t . On the o t h e r hand L i n d i g and Rodgers (12) r e p o r t e d that i n the presence of d e u t e r i u m o x i d e , which p r o l o n g s s i n g l e t oxygen l i f e t i m e s , the i n t e r f a c i a l r e g i o n c o u l d a f f e c t s i n g l e t lifetimes. With e i t h e r a n i o n i c or c a t i o n i c m i c e l l e s i n d e u t e r i u m oxide s i n g l e t l i f e t i m e was found to be 54 microseconds ( i . e . about

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ANIONIC MICELLES

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F i g u r e 4. S i n g l e t oxygen l i f e t i m e s i n d i f f e r e n t s o l v e n t systems. Adapted from R e f s . 12, 13, 14, and 62.

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1 3 - f o l d l o n g e r than i n w a t e r ) . With n o n i o n i c m i c e l l e s the l i f e t i m e was o n l y 21 to 26 m i c r o s e c o n d s . The r e d u c t i o n was a t t r i b u t e d to quenching by h y d r o x y l groups at the interfacial r e g i o n of the nonionic m i c e l l e s . A second study confirmed t h a t some, but not a l l , n o n - i o n i c m i c e l l e s produce s i m i l a r l i f e t i m e r e d u c t i o n s i n d e u t e r i u m oxide (49). The f u n c t i o n a l groups at the i n t e r f a c e may not be the o n l y way i n which s i n g l e t l i f e t i m e s are a f f e c t e d by t h i s boundary. Based on s t u d i e s i n a r e v e r s e m i c e l l a r system (aqueous aggregates i n a hydrophobic s o l v e n t ) , Miyoshi and Tomita (63) have proposed that s i n g l e t oxygen may not be s u b j e c t to quenching by s t r u c t u r e d water near membrane s u r f a c e s . Thus there would be no e f f e c t of deuterium oxide i n t h i s domain. A z i d e quenching of and tryptophan r e a c t i v i t y with s i n g l e t oxygen a l s o appear to be reduced i n t h i s domain ( 6 4 ) . The i s s u e of the i n t e r a c t i o n of s i n g l e t oxygen w i t h the membrane i n t e r f a c e r a i s e s an important q u e s t i o n . How w e l l can s i n g l e t oxygen d i f f u s e i n t o , out o f , o S i n g l e t Oxygen D i f f u s i o n a c r o s s Membranes. Oxygen d i f f u s e s v e r y easily through membranes and a l l b i o l o g i c a l membranes are quite permeable to oxygen. There i s no reason to suspect that e x c i t e d state oxygen should differ from the ground s t a t e in its diffusibility. I n f a c t a l l the c a l c u l a t i o n s of mean d i f f u s i o n d i s t a n c e s f o r s i n g l e t oxygen use the d i f f u s i o n c o e f f i c i e n t f o r ground s t a t e oxygen. Oxygen s o l u b i l i t y i s a c t u a l l y g r e a t e r i n hydrocarbon s o l v e n t s and m i c e l l e s than i n water ( 6 2 ) . On the o t h e r hand i t i s not o b v i o u s t h a t s i n g l e t oxygen, i n t r a v e r s i n g a membrane, w i l l remain i n the e x c i t e d s t a t e . Not only i s there a change i n medium p o l a r i t y upon e n t e r i n g the hydrocarbon c o r e , but the various f u n c t i o n a l groups a s s o c i a t e d w i t h the i n t e r f a c i a l r e g i o n must be penetrated. It i s now known that s i n g l e t oxygen can penetrate membranes under a p p r o p r i a t e c o n d i t i o n s . Gorman, L e v e r i n g and Rodgers (18) attacked t h i s problem by making SDS m i c e l l e s c o n t a i n i n g a reagent, diphenyl isobenzofuran (DPBF), which i s bleached specifically by s i n g l e t oxygen. To this suspension they added a sensitizer, methylene b l u e , which i s c o n f i n e d to the aqueous suspension medium. Upon i r r a d i a t i o n the DPBF was bleached by s i n g l e t oxygen which d i f f u s e d from the medium i n t o the m i c e l l e s . They f u r t h e r showed t h a t if they produced a second set of m i c e l l e s c o n t a i n i n g the lipids o l u b l e s e n s i t i z e r , pyrene, and added these to m i c e l l e s c o n t a i n i n g DPBF, s i n g l e t oxygen c o u l d d i f f u s e out of the pyrene c o n t a i n i n g m i c e l l e s where i t was generated and i n t o the DPBF m i c e l l e s to produce b l e a c h i n g , F i g u r e 5. Of p a r t i c u l a r note i s the f i n d i n g t h a t i n the methylene b l u e system the r a t e of DPBF b l e a c h i n g by s i n g l e t oxygen was reduced 50% by the c o m p a r t m e n t a l i z a t i o n . Thus s i n g l e t oxygen was a b l e to p e n e t r a t e i n t o the m i c e l l e s but an a p p r e c i a b l e fraction may have been quenched. Many s t u d i e s have s i n c e been performed u s i n g m i c e l l e s , r e v e r s e d m i c e l l e s and liposomes. Some s t u d i e s have shown e s s e n t i a l l y no h i n d r a n c e i n s i n g l e t oxygen p e n e t r a t i o n of the membrane model (65). Others have c l a i m e d no h i n d r a n c e but have actually shown a v a r i a b l e decrease i n r e a c t i o n r a t e (66 {5-22% i n h i b i t i o n > , 6 7 <12.5%>). However, a few s t u d i e s have shown significant i n t e r a c t i o n of the membrane w i t h p e n e t r a t i n g singlet

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F i g u r e 5. Diagram of s i n g l e t oxygen r e a c t i o n s i n a m i c e l l a r system. S i n g l e t oxygen g e n e r a t e d by p h o t o e x c i t e d pyrene can d i f f u s e out of the m i c e l l e i n which i t was produced. Three competing pathways e x i s t with d i f f e r i n g rate constants. Spontaneous d e e x c i t a t i o n to the ground s t a t e , k^, quenching by empty m i c e l l e s , kq, and e n t r y i n t o a DPBF-containmg m i c e l l e , k . B l e a c h i n g of DPBF by s i n g l e t oxygen i s f o l l o w e d s p e c t r o p h o t o m e t r i c a l l y . Adapted from R e f s . 18 and 63.

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oxygen. Miyoshi and Tomita (68) found that m i c e l l e s produced quenching of s i n g l e t oxygen as e f f i c i e n t l y as a z i d e . They estimated t h a t the p r o b a b i l i t y of s i n g l e t oxygen p e n e t r a t i o n g i v e n an encounter w i t h a m i c e l l e was about 0.38 to 0.48. Gorman, £t a l . (18) e s t i m a t e d t h i s value as o n l y 0.1. Suwa, et a l . (45) demonstrated that c h o l e s t e r o l i n c o r p o r a t e d i n t o m i c e l l e s was e f f i c i e n t l y photomodified only i f s e n s i t i z e r was a l s o i n the m i c e l l e , not i f i t was d i s s o l v e d in the suspension medium. F i n a l l y J o r i and co-workers (^9) found that s e n s i t i z e r s e p a r a t e d i n m i c e l l a r s o l u t i o n from i t s t a r g e t was a b l e to modify i t only under some c o n d i t i o n s . How can these d i v e r s e r e s u l t s be r e c o n c i l e d ? C e r t a i n l y the d i f f e r e n c e s i n m i c e l l e or liposome c o m p o s i t i o n , s e n s i t i z e r employed and t a r g e t can i n f l u e n c e the results. This i s r e f l e c t e d by the r e s u l t s d e s c r i b e d above i n which s i n g l e t oxygen l i f e t i m e s were reduced by n e u t r a l but not c a t i o n i c o r a n i o n i c m i c e l l e s i n a s i n g l e study (L2) • The c o n c l u s i o n seems t o be t h a t under some c o n d i t i o n s i n simple model systems s i n g l e t oxygen may p e n e t r a t instances there can b oxygen. The s i t u a t i o s r e m i n i s c e n t o the i s s u e o effective sensitizer location. A l t h o u g h s e n s i t i z e r e x t e r n a l t o the membrane may e f f e c t m o d i f i c a t i o n through s i n g l e t oxygen g e n e r a t i o n , in biological systems membrane a s s o c i a t e d s e n s i t i z e r i s the e f f e c t i v e species. So, here a l s o , the r e a l q u e s t i o n i s what i s the p e n e t r a b i l i t y of s i n g l e t oxygen f o r b i o l o g i c a l membranes? A l l of the model systems d i s c u s s e d a r e d e v o i d of p r o t e i n s . Membrane proteins are good t a r g e t s f o r r e a c t i o n w i t h s i n g l e t oxygen. Thus, s i g n i f i c a n t reduction i n s i n g l e t oxygen c o n c e n t r a t i o n s may o c c u r as i t passes into or through p r o t e i n - c o n t a i n i n g b i o l o g i c a l membranes. No e x p e r i m e n t a l evidence i s a v a i l a b l e c o n c e r n i n g t h i s p o i n t . E f f e c t s of Temperature and Membrane F l u i d i t y . Temperature e f f e c t s on membrane p h o t o m o d i f i c a t i o n appear to be d i v e r s e at f i r s t s i g h t . For photohemolysis by f l u o r e s c e i n d e r i v a t i v e s Blum, eit a l . , (70) showed almost no temperature dependence f o r the p h o t o m o d i f i c a t i o n process ( d u r i n g i l l u m i n a t i o n ) and Davson and Ponder (7^) showed that even the photodynamic l y s i s o c c u r i n g a f t e r l i g h t was r e l a t i v e l y independent of temperature. Blum and Kauzmann (72) were a b l e t o show t h a t a t severely reduced t e m p e r a t u r e s , -79 and -210 C, p h o t o h e m o l y t i c membrane m o d i f i c a t i o n was g r e a t l y reduced and a b o l i s h e d r e s p e c t i v e l y . On the o t h e r hand s e n s i t i z e r a s s o c i a t i o n w i t h the membrane varies d i r e c t l y with temperature i n the i n t e r v a l b e f o r e illumination ( P o o l e r , p e r s o n a l communication). I n yeast c e l l s p h o t o i n a c t i v a t i o n s e n s i t i z e d by t o l u i d e n e b l u e i s a c c e l e r a t e d at h i g h e r temperatures w i t h a break p o i n t at 21-22° C (73^). T h i s has been a t t r i b u t e d to a change i n membrane f l u i d i t y at the t r a n s i t i o n temperature of the membrane. Membranes have been shown t o a l t e r t h e i r dye p e r m e a b i l i t y a t the phase t r a n s i t i o n of the membrane l i p i d s ( 7 4 ) . I n liposomes a l s o there appears to be a d i f f e r e n c e i n p h o t o m o d i f i c a t i o n r a t e which i s dependent on the phase t r a n s i t i o n of the l i p i d . Suwa, e_t a l . (75) used two d i f f e r e n t l i p i d s w i t h d i f f e r e n t t r a n s i t i o n temperatures. Photomodification of c h o l e s t e r o l i n c o r p o r a t e d i n t o the liposomes was g r e a t l y i n c r e a s e d above the r e s p e c t i v e t r a n s i t i o n temperature of each type of liposome. The enhanced p h o t o m o d i f i c a t i o n was a s s o c i a t e d w i t h

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an enhanced uptake of the s e n s i t i z e r , hematoporphyrin. High c h o l e s t e r o l l e v e l s are known t o a b o l i s h phase t r a n s i t i o n s . With h i g h cholesterol l e v e l s (1:2, c h o l e s t e r o l : p h o s p h o l i p i d ) hematoporphyrin incorporation and c h o l e s t e r o l m o d i f i c a t i o n showed no abrupt a l t e r a t i o n w i t h temperature. The above f i n d i n g s are most c o n s i s t e n t w i t h a temperature dependence of s e n s i t i z e r a s s o c i a t i o n w i t h the membrane. I n most s t u d i e s i n which temperature was not v a r i e d u n t i l the time of i l l u m i n a t i o n or a f t e r , no temperature dependence was seen. When p r e illumination i n c u b a t i o n occured at d i f f e r e n t temperatures the temperature dependence was d e t e c t e d . Suwa, ejt a l . (75^), w h i l e r e c o g n i z i n g the importance of s e n s i t i z e r a s s o c i a t i o n proposed that t h i s c o u l d not e n t i r e l y account f o r the observed temperature dependence. They f e l t that i n a d d i t i o n to f a c i l i t a t i n g sensitizer a s s o c i a t i o n , the i n c r e a s e i n membrane f l u i d i t y w i t h temperature augmented p h o t o m o d i f i c a t i o n r a t e s by enhancing oxygen solubility. T h i s was based on the t h a t oxygen solubilit transition temperature bilayers y biologica membranes do not e x h i b i t w e l l d e f i n e d phase transitions the a p p l i c a b i l i t y of t h i s o b s e r v a t i o n to c e l l membranes i s u n c e r t a i n . Summary and

Conclusions

Membrane p h o t o m o d i f i c a t i o n and s i n g l e t oxygen g e n e r a t i o n i n membranes are o b v i o u s l y d i f f e r e n t from the analogous p r o c e s s e s i n simple homogenous s o l u t i o n . Membranes are s t r u c t u r e d , c o m p a r t m e n t a l i z e d systems of l i p i d s , p r o t e i n s and c h o l e s t e r o l w i t h domains of v a r y i n g h y d r o p h o b i c i t y and r e a c t i v i t y . The i n t e r a c t i o n of s e n s i t i z e r s with the membrane can be p i v o t a l in sensitization reactions. Both h a l o g e n a t e d f l u o r e s c e i n s and p o r p h y r i n s appear to l o c a l i z e near the membrane i n t e r f a c e and are e f f e c t i v e from that l o c a t i o n . They a r e relatively i n e f f e c t i v e , f o r m o d i f i c a t i o n of b i o l o g i c a l membranes, when g e n e r a t i n g s i n g l e t oxygen i n the medium e x t e r n a l to the membrane. S i n g l e t oxygen can modify many membrane components. Singlet oxygen quantum y i e l d s may be e i t h e r i n c r e a s e d or decreased i n the membrane environment depending on the sensitizer employed. P o r p h y r i n s are d i s a g g r e g a t e d by membrane a s s o c i a t i o n and demonstrate i n c r e a s e d quantum y i e l d s . Halogenated f l u o r e s c e i n s , which show no a g g r e g a t i o n e f f e c t s , have lower quantum y i e l d s i n membranes. S i n g l e t oxygen lifetimes are i n c r e a s e d i n the membrane environment independent of the mode of g e n e r a t i o n . I t can d i f f u s e a c r o s s membrane i n t e r f a c e s but the s i g n i f i c a n c e of t h i s in biological membranes i s q u e s t i o n a b l e . Finally temperature can modulate photomodification rates, p r o b a b l y through e f f e c t s on s e n s i t i z e r a s s o c i a t i o n w i t h the membrane and p o s s i b l y by i n c r e a s e d oxygen s o l u b i l i t y above the phase t r a n s i t i o n temperature of the membrane l i p i d s . [Note: For completeness the reader s h o u l d be aware that s e n s i t i z a t i o n by p s o r a l e n s has not been c o n s i d e r e d here. Psoralens a c t by n o n - s i n g l e t oxygen mechanisms on c e l l u l a r DNA. A c r i d i n e s and r e l a t e d s e n s i t i z e r s , which a l s o a f f e c t DNA, have l i k e w i s e not been t r e a t e d . ]

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A number of q u e s t i o n s c o n c e r n i n g membrane p h o t o m o d i f i c a t i o n remain unanswered. These i n c l u d e the f o l l o w i n g . 1. ) What i s ( a r e ) the membrane t a r g e t ( s ) which a r e c r i t i c a l f o r cell killing (inactivation, lysis)? 2. ) What i s t h e most e f f e c t i v e l o c a t i o n f o rsensitizer i n b i o l o g i c a l membranes? 3. ) What a r e the t r i p l e t quantum y i e l d s f o r halogenated fluoresceins and the s i n g l e t oxygen quantum y i e l d s i n membranes? 4. ) What i s t h e l i f e t i m e o f s i n g l e t oxygen i n b i o l o g i c a l membranes? 5. ) What i s the p e n e t r a b i l i t y of singlet oxygen through b i o l o g i c a l membranes? 6. ) Can s e n s i t i z e r uptake account f o r the temperature dependence of membrane p h o t o m o d i f i c a t i o n ? In c o n c l u s i o n membranes appear to be e x c e l l e n t targets f o r photomodification. Man the membrane, some generat oxygen s o l u b i l i t y and henc oxyge highe membrane i n t e r i o r , and s i n g l e t oxygen l i f e t i m e s a r e l o n g e r i n the membrane i n t e r i o r . Since many o f the m o l e c u l a r components o f membranes a r e s u s c e p t i b l e t o p h o t o m o d i f i c a t i o n r e a c t i o n s , conditions strongly favor membrane m o d i f i c a t i o n . Perhaps then i t is u n d e r s t a n d a b l e , as s t a t e d a t the o u t s e t o f t h i s c h a p t e r , that membranes are so o f t e n i d e n t i f i e d as c r i t i c a l t a r g e t s i n c e l l u l a r and organism p h o t o s e n s i t i z a t i o n . Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Bellnier, D.A.; Dougherty, T . J . Photochem. Photobiol. 1982, 36, 43-47. Kessel, D. Biochem. 1977, 16, 3443-3449. Moan, J.; Pettersen, E.O.; Christensen, T. Br. J. Cancer 1979, 39, 398-407. Kohn, K . ; Kessel, D. Biochem. Pharmacol. 1979, 28, 2465-2470. Volden, G.T.; Christensen, T.; Moan, J . Photobiochem. Photobiophys. 1981, 3, 105-111. Henderson, B.W.; Bellnier, D.A.; Ziring, B.; Dougherty, T . J . Adv. Exper. Med. Biol. 1983, 160, 129-138. Ehrenberg, B.; Malik, Z . ; Nitzan, Y. Photochem. Photobiol. 1985, 41, 429-435. Singer, S . J . ; Nicolson, G.L. Science 1972, 175, 720-731. Lux, S.; Shohet, S.B. Hospital Practice 1984, 19, 77-83. Shohet, S.B.; Lux, S. Hospital Practice 1984, 19, 89-108. Gilbert, D . L . ; Ehrenstein, G. Cur. Top. Membr. Transp. 1985, 22, 407-421. Lindig, B.A.; Rodgers, M.A.J. J . Phys. Chem. 1979, 83, 16831688. Rodgers, M.A.J.; Snowden, P.T. J. Amer. Chem. Soc. 1982, 104, 5543-5545. Parker, J . G . ; Stanbro, W.D. Porphyrin Localization and Treatment of Tumors.; Alan R. Liss, Inc.: 1984; pp 259-284. Pooler, J . P . ; Valenzeno, D.P. Photochem. Photobiol. 1979, 30, 581-584.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

56

16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48.

LIGHT-ACTIVATED PESTICIDES

Lindig, B.A.; Rodgers, M.A.J. Photochem. Photobiol. 1981, 33, 627-634. Bezman, S.A.; Burtis, P.A.; Izod, T . P . J . ; Thayer, M.A. Photochem. Photobiol. 1978, 28, 325. Gorman, A . A . ; Lovering, G . ; Rodgers, M.A.J. Photochem. Photobiol. 1976, 23, 399-403. Eisenberg, W.C.; Taylor, K.; Grossweiner, L . I . Photochem. Photobiol. 1984, 40, 55-58. Sandberg, S.; Glette, J.; Hopen, G.; Solberg, C.O.; Romslo, I. Photochem. Photobiol. 1981, 34, 471-475. Pooler, J . P . ; Valenzeno, D.P. Photochem. Photobiol. 1979, 30, 491-498. Valenzeno, D.P.; Pooler, J . P . Photochem. Photobiol. 1982, 35, 343-350. Pooler, J . P . ; Girotti, A.W. hotochem. Photobiol.1986, 44, 495499. Pooler, J . P . Photochem Cook, J . S . ; Blum, H.F Comp Physiol , , Valenzeno, D.P. Photochem. Photobiol. 1984, 40, 681-688. Valenzeno, D.P. I.E.E.E. J . Quant. Electronics 1984, QE20, 14391441. Waggoner, A. J. Membr. Biol. 1976, 27, 317-334. Easton, T.G.; Valinsky, J.E.; Reich, E. Cell 1978, 13, 475-486. Valinsky, J.E.; Easton, T.G.; Reich E. Cell 1978, 487-499. Schlegel, R.A.; Phelps, B.V.; Waggoner, A . ; Terada, L . ; Williamson, P. Cell 1980, 20, 321-328. Kalyanaraman, B.; Sieber, F. Photochem. Photobiol. 1986, 43, 28s. Lelkes, P . I . ; Miller, I.R. J . Membr. Biol. 1980, 52, 1-15. Bagchi, B.; Basu, S. Photochem. Photobiol. 1979, 29, 403-405. Pooler, J . P . ; Valenzeno, D.P. Photochem. Photobiol. 1978, 28, 219-228. Varnadore, W.E.; Arrieta, R.T.; Duchek, J . R . ; Huebner, J . S . J. Membr. Biol. 1982, 65, 147-153. Floyd, R.A. Biochem. Biophys. Res. Commun. 1980, 96, 1305-1311. Rodgers, M.A.J. Chem. Phys. Lett. 1981, 78, 509-514. Rodgers, M.A.J. J . Phys. Chem. 1981, 85, 3372-3374. Minch, M.J.; Shah, S.S. J. Org. Chem. 1979, 44, 3252-3255. Wade, M . J . ; Spikes, J.D. Photochem. Photobiol. 1971, 14, 221224. Emiliani, C.; Delmelle, M. Photochem. Photobiol. 1983, 37, 487490. Sandberg, S.; Romslo, I. Clin. Chim. Acta 1981, 109, 193-201. Kessel, D.; Kohn, K.I. Cancer Res. 1980, 40, 303-307. Suwa, K.; Kimura, T . ; Schaap, A.P. Biochem. Biophys. Res. Commun. 1977, 75, 785-792. Moan, J.; Sommer, S. Photochem. Photobiol. 1984, 40, 631-634. Dougherty, T . J . ; Boyle, D.G.; Weishaupt, K.R.; Henderson, B.A.; Potter, W.R.; Bellnier, D.A.; Wityk, K.E. Adv. Exptl. Med. Biol. 1983, 160, 3-13. Pryor, W.A. Photochem. Photobiol. 1978, 28, 787-801. Deziel, M.R.; Girotti, A.W. J. Biol. Chem. 1980, 255, 8192-8198.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

3. VALENZENO 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75.

Photomodification and Singlet Oxygen Generation

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Rodgers, M.A.J. Photochem. Photobiol. 1983, 37, 99-103. Girotti, A.W. J. Biol. Chem. 1978, 253, 7186-7193. Deziel, M.R.; Girotti, A.W. Photochem. Photobiol. 1980, 31, 593596. Reyftman, J . P . ; Santus, R.; Moliere, P.; Kohen, E. Photobiochem. Photobiophys. 1985, 9, 183-192. Blum, A; Grossweiner, L . I . Photochem. Photobiol. 1985, 41, 2732. Murasecco, P.; Oliverso, E . ; Braun, A.M.; Monnier, P. Photobiochem. Photobiophys. 1985, 9, 193-201. Keene, J . P . ; Kessel, D.; Land, E.J.; Redmond, R.W.; Truscott, T.G. Photochem. Photobiol. 1986, 43, 117-120. Reddi, E . ; Jori, G.; Rodgers, M.A.J.; Spikes, J.D. Photochem. Photobiol. 1983, 38, 639-645. Gandin, E . ; Lion, Y . ; Van de Vorst, A. Photochem. Photobiol. 1983, 37, 271-278. Fleming, G.R.; Knight Robinson, G.W. J. Amer McClure, D.S.; Blake, N.W.; Hanst, P.L. J. Chem. Phys. 1954, 22, 255-258. Bowers, P.G.; Porter, G. Proc. Roy Soc. (Lond.) 1967, 296A, 435441. Reddi, E . ; Rodgers, M.A.J.; Spikes, J . D . ; Jori, G. Photochem. Photobiol. 1984, 40, 415-421. Lee, P.C.; Rodgers, M.A.J. J . Phys. Chem. 1983, 87, 4894-4898. Miyoshi, N.; Tomita, G. Z. Naturforsch. 1979, 34b, 339-343. Rodgers, M.A.J.; Lee, P.C. J. Phys. Chem. 1984, 88, 3480-3484. Gorman, A.A.; Rodgers, M.A.J.; Chem. Phys. Lett. 1978, 55, 5254. Kraljic, I . ; Barboy, N.; Leicknam, J . P . Photochem. Photobiol. 1979, 30, 631-633. Matheson, I.B.C.; Lee, J.; King, A.D. Chem. Phys. Lett. 1978, 55, 49-51. Miyoshi, N.; Tomita, G. Z. Naturforsch. 1978, 33b, 622-627. Sconfienza, C.; Van de Vorst, A . ; Jori, G. Photochem. Photobiol. 1980, 31, 351-357. Blum, H . F . ; Pace, N.; Garrett, R.L. J. Cell. Comp. Physiol. 1937, 9, 217-228. Davson, H . ; Ponder, E. J. Cell. Comp. Physiol. 1940, 67-74. Blum, H . F . ; Kauzmann, E.F. J. Gen. Physiol. 1954, 37, 301-311. Ito, T. Photochem. Photobiol. 1981, 33, 117-120. Braganza, L . F . ; Blott, B.H.; Coe, T.J.; Melville, D. Biochim. Biophys. Acta 1983, 731, 137-144. Suwa, K.; Kimura, T . ; Schaap, A.P. Photochem. Photobiol. 1978, 28, 469-473.

RECEIVED November 20, 1986

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 4

Identifying Singlet Oxygen in Chemical, Photochemical, and Enzymic Reactions Ahsan U. Khan Department of Chemistry, Harvard University, Cambridge, MA 02138 and Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306

Application of ultrasensitiv spectroscopy to studie at ambient temperatur produce unambiguou results. From photosensitization comes evidence of singlet oxygen generation by dyes in solvents, including H O ; quenching of singlet oxygen by vitamin C; and of singlet oxygen-solvent interaction. The tetracyclines show direct correlation between the efficiency of singlet oxygen generation and their clinical phototoxicity. Biological singlet oxygen, the observation of enzyme systems generating singlet oxygen, was found for the peroxidases — myeloperoxidase, lactoperoxidase and chloroperoxidase. Lipoxygenase exhibits a weak singlet oxygen luminescence. Spectral evidence of singlet oxygen generation in the thermal dissociation of the polycyclic endoperoxides is now available. A highly efficient low-temperature source of singlet oxygen was discovered in the reaction of triethylsilane with ozone. 2

In c u r r e n t c h e m i c a l , p h o t o c h e m i c a l and b i o l o g i c a l r e s e a r c h , s i n g l e t oxygen i s o f t e n p r o p o s e d as t h e r e a c t i v e i n t e r m e d i a t e (1-2). The t r a n s i e n t p r e s e n c e o f s i n g l e t oxygen i s g e n e r a l l y deduced from c h e m i c a l p r o d u c t s , s c a v e n g e r t r a p p i n g and o t h e r s e c o n d a r y e v i d e n c e . Many o f t h e s e s e c o n d a r y e f f e c t s can e q u a l l y i n d i c a t e t h e p r e s e n c e of o t h e r r e a c t i v e i n t e r m e d i a t e s — C^**/ OH«, HOO" — and a l s o c a n not d i s t i n g u i s h between sigma and d e l t a s i n g l e t oxygen. An unambiguous i d e n t i f i c a t i o n o f s i n g l e t (^Ag) oxygen m o l e c u l e s i n s o l u t i o n i s c r u c i a l t o t h e growth o f t h i s r e s e a r c h f i e l d . Over t h e l a s t number o f y e a r s we have d e v e l o p e d u l t r a s e n s i t i v e s p e c t r o p h o t o m e t e r s f o r t h e n e a r i n f r a r e d , i n i t i a l l y b a s e d on a thermoelectrically cooled lead sulfide detector, optimized optics, i n t e g r a t o r s and d a t a p r o c e s s o r s Q ) , l a t e r more s e n s i t i v e i n s t r u m e n t s b a s e d on a germanium d e t e c t o r (A)• The p r e s e n t s p e c t r o m e t e r c o v e r s t h e range o f 1.0 t o 1.7 m i c r o n , and i s c a p a b l e 5

of

d e t e c t i n g b o t h t h e (0,0) and (0,1)

1

A

oxygen m o l e c u l e a t 12 68 nm and 1586 nm,

3

g

~* Z g t r a n s i t i o n s o f t h e respectively.

0097-6156/87/0339-0058$06.00/0 © 1987 American Chemical Society

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

4.

KHAN

Identifying Singlet Oxygen

59

In 197 6 K r a s n o v s k y (£) and i n 1978 B y t e v a and G u r i n o v i t c h (1) were a b l e t o o b s e r v e t h e p h o t o s e n s i t i z e d 1270 nm e m i s s i o n o f s i n g l e t oxygen i n CC1 solution. In CC1 s i n g l e t oxygen has one o f t h e 4

4

longest observed s o l u t i o n l i f e t i m e s . We have used t h e h i g h s e n s i t i v i t y l u m i n e s c e n c e s p e c t r o m e t e r t o compare s i n g l e t oxygen emission c h a r a c t e r i s t i c s i n d i f f e r e n t s o l v e n t s , t o f o l l o w the k i n e t i c s o f r e a c t i o n s o f s i n g l e t oxygen, t o d i s c r i m i n a t e between m e c h a n i s t i c a l t e r n a t i v e s , and t o d i s c o v e r new c h e m i c a l and b i o l o g i c a l s o u r c e s o f s i n g l e t oxygen. A l t h o u g h v i s i b l e e m i s s i o n spectroscopy p l a y e d a c r i t i c a l r o l e i n the d i s c o v e r y of the c h e m i c a l g e n e r a t i o n (2) and t h e subsequent c h a r a c t e r i z a t i o n o f s i n g l e t oxygen i n t h e r e d c h e m i l u m i n e s c e n c e o f hydrogen p e r o x i d e h y p o c h l o r i t e r e a c t i o n (8-9), t h i s p a p e r i s o n l y c o n c e r n e d w i t h i n f r a r e d emission. T h i s p r e s e n t a t i o n has t h e f o l l o w i n g o u t l i n e : (1) a b r i e f d e s c r i p t i o n o f t h e l a t e s t v e r s i o n o f t h e l u m i n e s c e n c e s p e c t r o m e t e r ; ( l i ) e l e c t r o n i c energy t r a n s f e r g e n e r a t i o n o f s i n g l e t oxygen i n a) s p e c t r o s c o p y o f d i s s o l v e d oxygen, b) p h o t o s e n s i t i z a t i o n by dye f biological interest ) kinetic f s i n g l e t oxygen r e a c t i o by d r u g s ; ( H i ) enzymi m i c r o b i c i d a l enzymes - m y e l o p e r o x i d a s e and l a c t o p e r o x i d a s e , b) p l a n t enzymes - c h l o r o p e r o x i d a s e c) b i o s y n t h e t i c enzymes l i p o x y g e n a s e ; (ly_) t h e r m a l g e n e r a t i o n ; and (;y_) a new s o u r c e o f chemical generation. Instrumentation In F i g u r e 1 i s shown the h i g h s e n s i t i v i t y l u m i n e s c e n c e s p e c t r o m e t e r . The s p e c t r o m e t e r c o n s i s t s o f a Spex Minimate I I , f/4.0 monochromator (Spex I n d u s t r i e s , Metuchen, N . J . ) , f i t t e d w i t h a 1.25 m i c r o n b l a z e d g r a t i n g , l i q u i d n i t r o g e n c o o l e d germanium d e t e c t o r 403L ( A p p l i e d D e t e c t o r , F r e s n o , CA), f o l l o w e d by a low n o i s e a m p l i f i e r PAR model 113 (E.G.&G. P r i n c e t o n A p p l i e d Research, P r i n c e t o n , N . J . ) , l o c k - i n a m p l i f i e r (PAR model 5207), l e a d i n g t o a Spex Datamate w i t h d i g i t a l s t o r a g e and p r i n t o u t . An o p t i c a l f i l t e r , ( F ) , C o r n i n g CS 7-56 i s p l a c e d b e f o r e t h e e n t r a n c e s l i t o f t h e monochromator t o r e j e c t second o r d e r i n t e r f e r i n g e m i s s i o n s . A c o l l e c t i n g l e n s , ( L ) , f o c u s e s t h e monochromator o u t p u t onto t h e detector crystal. The e s t i m a t e d s e n s i t i v i t y o f t h e l u m i n e s c e n c e s p e c t r o m e t e r i s 10** photons p e r second a t 1270 nm. The e s t i m a t e i s b a s e d on t h e assumption t h a t t h e t h e r m a l d i s s o c i a t i o n o f 1,4-dimethyl X

naphthalene-1,4-endoperoxide l e a d s t o a 100% y i e l d o f 0 ( A ) (10H) i n carbon t e t r a c h l o r i d e s o l u t i o n a t 50°C. The assumed l i f e t i m e i n c a r b o n t e t r a c h l o r i d e i s 20 msec a t t h i s t e m p e r a t u r e (.12.) . 2

E l e c t r o n i c Energy T r a n s f e r G e n e r a t i o n

of S i n g l e t

g

Oxygen

The f i r s t i n d i c a t i o n t h a t a l i g h t - d e p e n d e n t a c t i v a t i o n might e x i s t f o r m o l e c u l a r oxygen was t h e d i s c o v e r y o f t h e spontaneous o x i d a t i o n of naphthacene i n t h e p r e s e n c e o f l i g h t and a i r by F r i t z s c h e i n 1867 (12), f o l l o w e d by t h e d i s c o v e r y by Raab i n 1900 (14) o f damage t o l i v i n g t i s s u e by t h e s y n e r g i s t i c e f f e c t o f l i g h t , a i r and o r g a n i c dye m o l e c u l e s . Both t h e s e e f f e c t s a r e now r e c o g n i z e d as examples o f p h o t o o x i d a t i o n r e a c t i o n s . Much i n t e r e s t c e n t e r e d on these r e a c t i o n s i n the e a r l y p a r t of t h i s century, r e s u l t i n g i n t h e i r c h a r a c t e r i z a t i o n by p r o d u c t i s o l a t i o n and i d e n t i f i c a t i o n and kinetic analysis. Kautsky and de B r u i j n i n 1931 (1£) s p e c u l a t e d

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987. ADC 403L J

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61

Identifying Singlet Oxygen

KHAN

t h a t p h o t o o x i d a t i o n mechanisms i n v o l v e d s i n g l e t oxygen. However, by t h e l a t e 1 9 3 0 ' s K a u t s k y ' s s p e c u l a t i o n s were b u r i e d under s e v e r e c r i t i c i s m by h i s c o n t e m p o r a r i e s ( 1 6 - 1 8 ) . The r e c o g n i t i o n of s i n g l e t oxygen as a c h e m i c a l s p e c i e s came w i t h t h e r e c o g n i t i o n of the c h e m i c a l g e n e r a t i o n o f s i n g l e t oxygen i n the s i m p l e c h e m i c a l r e a c t i o n o f h y p o c h l o r i t e i o n w i t h hydrogen p e r o x i d e i n t h e s p e c t r o s c o p i c d i s c o v e r y i n 1963 by Khan and Kasha ( 2 ) . R e e x a m i n a t i o n o f s i n g l e t oxygen as a r e a c t i v e i n t e r m e d i a t e i n p h o t o o x i d a t i o n r e a c t i o n s ensued ( 1 9 - 2 1 ) . D i r e c t a b s o r p t i o n o f l i g h t t o g e n e r a t e s i n g l e t oxygen i s h i g h l y i m p r o b a b l e because o f the low o s c i l l a t o r s t r e n g t h o f t r a n s i t i o n between t h e ground t r i p l e t s t a t e o f t h e oxygen m o l e c u l e and i t s f i r s t two e x c i t e d s i n g l e t s t a t e s ( 2 2 - 2 3 ) . Kawaoka, Khan and Kearns ( 2 4 - 2 5 ) i n 1967 e s t a b l i s h e d the t h e o r e t i c a l b a s i s o f p h o t o s e n s i t i z e d g e n e r a t i o n o f s i n g l e t oxygen i n t h e q u e n c h i n g o f organic t r i p l e t states. T h i s e l e c t r o n i c energy t r a n s f e r p r o c e s s c i r c u m v e n t s the r e s t r i c t i o n o f d i r e c t o p t i c a l e x c i t a t i o n and i s the h i g h l y e f f i c i e n t proces photooxidation reactions e n e r g y t r a n s f e r g e n e r a t i o n o f s i n g l e t oxygen i n t h e gas phase f o l l o w e d ( 2 6 - 2 8 ) . The f i r s t d i r e c t s p e c t r o s c o p i c o b s e r v a t i o n of s e n s i t i z e d g e n e r a t i o n of s i n g l e t oxygen i n s o l u t i o n , d e t e c t e d by t h e 12 68 nm n e a r i n f r a r e d e m i s s i o n , i s by K r a s n o v s k y (jj.) . He u s e d p h o t o m u l t i p l i e r d e t e c t i o n , C C I 4 as s o l v e n t , and c h l o r o p h y l l and r e l a t e d dyes as s e n s i t i z e r s . Khan and Kasha Q) d e v e l o p e d an u l t r a s e n s i t i v e n e a r i n f r a r e d s p e c t r o s c o p y and a p p l i e d i t t o s t u d y s i n g l e t (-^Ag) oxygen i n s o l u t i o n .

S p e c t r o s c o p y o f D i s s o l v e d Oxygen M o l e c u l e s : Oo (^&g) ••-Solvent Interaction. The e l e c t r o n i c s t a t e s o f m o l e c u l a r oxygen i n s o l u t i o n are t h e f o c u s o f t h i s p r e s e n t a t i o n . S p e c t r o s c o p i c i n v e s t i g a t i o n of m o l e c u l a r oxygen i n s o l u t i o n i n d i c a t e s v e r y l i t t l e , i f any, f r e q u e n c y s h i f t from the gas phase l u m i n e s c e n c e f r e q u e n c y o f s i n g l e t (^-Ag) s t a t e o f 0 ( 3 - 4 . 2 9 ) . However, u s i n g t h e Ge-based s p e c t r o m e t e r , Chou and Khan (.20.) o b s e r v e d d i s t i n c t new e m i s s i o n bands from oxygen s a t u r a t e d C C I 4 , C D C I 3 , C 2 F 3 C I 3 and C I Q 1 8 These new bands are much weaker ( r a t i o ~ 1/300 t o ~ 1/550) and r e d s h i f t e d from t h e (0,0) v i b r o n i c band o f A -> Z~. The appearance o f t h e s e bands i s c o n s i s t e n t w i t h a s i m u l t a n e o u s e l e c t r o n i c v i b r a t i o n a l t r a n s i t i o n i n v o l v i n g the A s t a t e o f oxygen and a v i b r a t i o n a l s t a t e of the s o l v e n t m o l e c u l e : 2

F

1

3

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The s p e c t r u m i n F i g u r e 2 shows c l e a r l y the s i n g l e t oxygen w i t h s o l v e n t m o l e c u l e s .

-> (solvent:

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P h o t o s e n s i t i z e d S i n g l e t Oxygen E m i s s i o n by Dyes o f B i o l o g i c a l Interest i n Liquid Solutions. Methylene b l u e s e n s i t i z e d g e n e r a t i o n o f s i n g l e t oxygen i n aqueous s o l u t i o n i s a commonly u s e d system f o r s t u d y i n g the p h o t o c h e m i c a l (31) and p h o t o b i o l o g i c a l (22.) e f f e c t s of t h e oxygen m o l e c u l e . From s e c o n d a r y e v i d e n c e i t was b e l i e v e d t h a t

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

1.20

1.30

1.40

1.50

1.60

1.40 1.50

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WAVELENGTH. MICRON

F i g u r e 2. P h o t o s e n s i t i z e d e m i s s i o n of d i s s o l v e d m o l e c u l a r oxygen at room temperature, broad band e x c i t a t i o n , 320-485 nm. (a) S o l v e n t C C l ^ , s e n s i t i z e r benzophenone, oxygen gas s a t u r a t e d . Spectrum d i s p l a y s the normal (0,0) and (0,1) e m i s s i o n s a t 1.28 micrometer and 1.58 micrometer, r e s p e c t i v e l y . New e m i s s i o n band appears at 1.42 micrometer. The i n s e r t d i s p l a y s the new band at t e n times e x p a n s i o n . (b) S o l v e n t CDCl^, s e n s i t i z e r perfluorobenzophenone, oxygen s a t u r a t e d . New e m i s s i o n band a t 1.42 micrometer. (Adapted from Reference 30.)

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

4. KHAN

Identifying Singlet Oxygen

63

>-

CO

C/) CO LU

5

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1.40

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WAVELENGTH, MICRON

Figure 2.—Continued. (c) Solvent C ^ C l ^ , s e n s i t i z e r p e r f l u o r o benzophenone, oxygen s a t u r a t e d . Spectrum shows two new e m i s s i o n bands at 1.42 micrometer and 1.49 micrometer. (d) S o l v e n t C F , s e n s i t i z e r perfluorobenzophenone, oxygen s a t u r a t e d . Spectrum shows the new e m i s s i o n band a t 1.49 micrometer, (Adapted from Reference 30.) 1 0

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

1 8

64

LIGHT-ACTIVATED PESTICIDES

s i n g l e t oxygen i s e f f i c i e n t l y g e n e r a t e d i n t h i s system, b u t , b e c a u s e o f i t s e x t r e m e l y s h o r t l i f e t i m e i n water (22.) , d i r e c t s i n g l e t oxygen e m i s s i o n was not o b s e r v a b l e . The i n a b i l i t y t o o b s e r v e s i n g l e t oxygen e m i s s i o n i n aqueous media r a i s e d q u e s t i o n s i n e v a l u a t i n g t h i s r e s e a r c h . U s i n g our s p e c t r o m e t e r b a s e d on t h e PbS d e t e c t o r , we o b s e r v e d t h e e m i s s i o n shown i n F i g u r e 3a. The 12 68 nm e m i s s i o n o f s i n g l e t oxygen i s s u p e r i m p o s e d on t h e t a i l o f the methylene b l u e e m i s s i o n . T h i s i s the f i r s t s p e c t r a l evidence o f s i n g l e t oxygen g e n e r a t i o n i n aqueous s o l u t i o n . Note t h a t i n t h e c h e m i l u m i n e s c e n c e o f hydrogen p e r o x i d e - h y p o c h l o r i t e r e a c t i o n i n aqueous s o l u t i o n , t h e e m i s s i o n o r i g i n a t e s i n t h e gas phase i n s i d e r e a c t i o n b u b b l e s (34). H e m a t o p o r p h y r i n s e n s i t i z e d g e n e r a t i o n o f s i n g l e t oxygen ( F i g u r e 3b) i s an e s p e c i a l l y i n t e r e s t i n g example because t h i s photodynamic pigment i s now u s e d w i t h s u c c e s s i n t h e p h o t o t h e r a p y o f c a n c e r v i a an a p p a r e n t s i n g l e t oxygen mechanism (2Zl) • I n j e c t e d i n t o t h e b l o o d s t r e a m , t h e pigment p r e f e r e n t i a l l adsorb th tumo d i photoexcited using fibe Krasnovsky (2£.) has r e p o r t e e m i s s i o n i n C C I 4 u s i n g v a r i o u s o t h e r p o r p h y r i n s as photosensitizers. 3 4 - b e n z p y r e n e an a t m o s p h e r i c p o l l u t a n t and a n o t o r i o u s c a r c i n o g e n i c agent (37-38), i s a l s o a p h o t o s e n s i t i z e r o f s i n g l e t oxygen, as shown i n F i g u r e 3c. f

f

K i n e t i c s o f S i n g l e t Oxygen R e a c t i o n i n Aqueous S o l u t i o n : Vitamin C Quenching o f S i n g l e t Oxygen.(39) L - a s c o r b i c a c i d , an aqueous phase a n t i o x i d a n t i n b o t h p l a n t and a n i m a l p h y s i o l o g y i s p r e s e n t i n a l l e u c a r y o t i c organisms (40-42) and i s a t o p i c o f l i v e l y i n t e r e s t b o t h i n c h e m i s t r y and m e d i c i n e i n r e c e n t t i m e s (A2). Chou and Khan s y n t h e s i z e d a w a t e r - s o l u b l e p h o t o s e n s i t z e r , c h r y s e n e sodium s u l f o n a t e , t o p h o t o s e n s i t i z e s i n g l e t oxygen i n aqueous s o l u t i o n . The q u e n c h i n g o f s i n g l e t oxygen by v i t a m i n C was s t u d i e d by d i r e c t l y m o n i t o r i n g t h e 12 68 nm e m i s s i o n . On comparing q u e n c h i n g o f p h o t o g e n e r a t e d s i n g l e t oxygen i n H 2 O and D 0 s o l u t i o n s , a marked 2

=

i s o t o p e e f f e c t was seen. Stern-Volmer constants are K ° 8-30 x 10 and K Q = 2.50 x 1 0 M " S " . The i s o t o p e e f f e c t p o i n t s t o a c h e m i c a l q u e n c h i n g o f s i n g l e t oxygen by v i t a m i n C, p o s s i b l y by f l at om a b s t r a c t i o n . F i g u r e s 4 & 5 summarize t h e r e s u l t s . Note t h a t t h e c h r y s e n e sodium s u l f o n a t e s e n s i t i z e d s i n g l e t oxygen e m i s s i o n s p e c t r u m i n aqueous medium does not have any o v e r l a p p i n g e m i s s i o n from t h e s e n s i t i z e r , compare w i t h t h e m e t h y l e n e b l u e spectrum, F i g u r e 3a. H 2 o

6

6

1

1

d 2 0

P h o t o s e n s i t i z a t i o n by Drugs: P h o t o t o x i c i t y of the T e t r a c y c l i n e s . T e t r a c y c l i n e s a r e one o f t h e most f r e q u e n t l y p r e s c r i b e d group o f a n t i b i o t i c s , d e r i v i n g t h e i r b a c t e r i o s t a t i c e f f e c t by p r e v e n t i n g t h e b i n d i n g o f aminoacyl-tRNA t o t h e a m i n o a c y l (A) s i t e o f t h e ribosome (AA)• A w e l l known s i d e e f f e c t o f t e t r a c y c l i n e t h e r a p y i s cutaneous p h o t o t o x i c i t y . C l i n i c a l estimates of p h o t o t o x i c i t y i n a s e r i e s of t e t r a c y c l i n e s c l e a r l y i n d i c a t e s that c h l o r o - d e r i v a t i v e s ( c h l o r o t e t r a c y c l i n e and d e m e c l o c y c l i n e ) a r e t h e most p h o t o t o x i c , t e t r a c y c l i n e i t s e l f b e i n g l e s s so, and m i n o c y c l i n e h a v i n g no a s s o c i a t e d p h o t o t o x i c i t y (45-49). In v i t r o c h e m i c a l s t u d i e s o f t e t r a c y c l i n e p h o t o s e n s i t i z a t i o n have s u g g e s t e d t h a t s i n g l e t oxygen i s t h e r e a c t i v e i n t e r m e d i a t e b e i n g g e n e r a t e d (50-53). Recently

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Identifying Singlet Oxygen

KHAN

1600

1200

800

1600

800

1200

1600

1200

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WAVELENGTH (nm) Figure

3.

1

3

Dye p h o t o s e n s i t i z e d (0,0) A -> Z " e m i s s i o n o f oxygen a temperature S e n s i t i z e r , methylen , , s a t u r a t e d , (b) S e n s i t i z e r h e m a t o p o r p h y r i n , s o l v e n t CCI4, 0 saturated. (c) S e n s i t i z e r 3,4-benzpyrene, solvent C C I 4 , 0 s a t u r a t e d . (Adapted from Ref. 3 ) . g

2

2

1.20 1.30 1.40 WAVELENGTH, MICRON Figure

4.

(a)

(0,0)

1

A

3

g

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1

oxygen i n [ H ] H 0 , s e n s i t i z e d by sodium 2

chrysene

3

s u l f o n a t e ( 1 0 ~ M), e x c i t a t i o n 320-485 nm, a t room temperature. (b) T o t a l q u e n c h i n g o f t h e 1.28 m i c r o n e m i s s i o n on a d d i t i o n o f L - a s c o r b i c a c i d (0.20 M). (Taken from Ref. 3 9 ) .

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

LIGHT-ACTIVATED PESTICIDES

66

Hasan and Khan (.5_4J d i r e c t l y m o n i t o r e d t h e p h o t o s e n s i t i z e d g e n e r a t i o n o f s i n g l e t oxygen i n t h e t e t r a c y c l i n e s e r i e s , d e m e c l o c y c l i n e , t e t r a c y c l i n e and m i n o c y c l i n e . They f o u n d o n e - t o one c o r r e s p o n d e n c e between t h e e f f i c i e n c y o f t h e s i n g l e t oxygen g e n e r a t i o n and t h e p h o t o t o x i c i t y o f t h e a n t i b i o t i c . The t e n t a t i v e c o n c l u s i o n i s t h a t s i n g l e t oxygen i s t h e r e a c t i v e i n t e r m e d i a t e i n the p h o t o t o x i c i t y of t e t r a c y c l i n e s . Figure 6 presents these findings. Enzymatic

Generation of S i n g l e t

Oxygen

A f t e r t h e c h e m i c a l d i s c o v e r y o f s i n g l e t oxygen, many attempts were made t o i m p l i c a t e s i n g l e t oxygen i n b i o l o g i c a l and b i o c h e m i c a l reactions. The main t o o l s used were m o n i t o r o f u l t r a w e a k v i s i b l e l u m i n e s c e n c e , c h e m i c a l p r o d u c t and c h e m i c a l s c a v e n g e r t e c h n i q u e s , and d e u t e r i u m k i n e t i c e f f e c t s . A l t h o u g h t h e s e t e c h n i q u e s a r e nons p e c i f i c i n d e t e c t i n g s i n g l e t oxygen, a number o f v a l u a b l e s u g g e s t i o n s emerged. K r i s h n a m u r t y and Simpson (5Ji) were t h e f i r s t to suggest the p o s s i b l r e a c t i o n s , u s i n g t h e fungus f l a v u s p r o d u c e s t h e enzyme q u e r c i t i n a s e t h a t o x i d i z e s q u e r c e t i n t o give a depside clevage product. Matsuura, e t a l (,5_£) had e a r l i e r o b t a i n e d t h e same d e p s i d e f o l l o w i n g p h o t o s e n s i t i z e d o x i d a t i o n , p r e s u m a b l y a s i n g l e t oxygen m e d i a t e d r e a c t i o n . K r i s h n a m u r t y and Simpson c o n c l u d e d t h a t s i n g l e t oxygen was t h e r e a c t i v e s p e c i e s e n z y m a t i c a l l y g e n e r a t e d by q u e r c i t i n a s e . Another important s u g g e s t i o n , due t o A l l e n e t a l and b a s e d on t h e o b s e r v a t i o n o f u l t r a w e a k v i s i b l e chemiluminescence, s u g g e s t e d t h a t s i n g l e t oxygen might be a p r o d u c t o f t h e m e t a b o l i s m o f p h a g o c y t o s i n g p o l y m o r p h o n u c l e a r l e u k o c y t e s (5JZ.) . These s u g g e s t i o n s r e s u l t e d i n an e x t e n s i v e s e a r c h f o r s i n g l e t oxygen i n enzymic and b i o l o g i c a l p r o c e s s e s but no c l e a r e v i d e n c e o f s i n g l e t oxygen g e n e r a t i o n emerged e i t h e r i n b i o l o g y o r i n b i o c h e m i s t r y . r

U s i n g t h e Ge b a s e d s p e c t r o m e t e r , Khan, Gebauer and Hager (5JJ.) p u b l i s h e d t h e f i r s t spectrum o f s i n g l e t oxygen e m i s s i o n from an enzymic r e a c t i o n , t h e c h l o r o p e r o x i d a s e / H 2 0 2 / C l " system, p r o v i d i n g i n c o n t r o v e r t i b l e e v i d e n c e o f s i n g l e t oxygen g e n e r a t i o n i n an enzymic system. Kanofsky (jjL£) has a l s o s t u d i e d enzymic g e n e r a t i o n of s i n g l e t oxygen i n t h i s system and o t h e r s w i t h a k i n e t i c a p p a r a t u s b a s e d on a Ge d e t e c t o r by m o n i t o r i n g 1270 nm e m i s s i o n using interference f i l t e r s . M i c r o b i c i d a l Enzymes: M y e l o p e r o x i d a s e : Polymorphonuclear Leucocytes. P r o b a b l y t h e most s i g n i f i c a n t enzyme o f p o l y m o r p h o n u c l e a r l e u c o c y t e s (PMN) i n v o l v e d i n t h e p h y s i o l o g i c a l defense a g a i n s t f o r e i g n bodies i s myeloperoxidase (MPO). MPO was o r i g i n a l l y i s o l a t e d by Agner and i s e s t i m a t e d t o c o n s t i t u t e g r e a t e r t h a n 5% o f t h e d r y weight o f t h e human PMN (£SL) • M a i n l y t h r o u g h t h e p i o n e e r i n g work o f K l e b a n o f f 161), t h e p o t e n t a n t i m i c r o b i a l system o f M P O / H 0 / h a l i d e was c h a r a c t e r i z e d . The M P O / H 0 / h a l i d e a n t i m i c r o b a l system i s t o x i c t o a wide v a r i e t y o f o r g a n i s m s : b a c t e r i a (61-62) , f u n g i (£2.) , v i r u s e s (££) , mycoplasma (££) , c h l y m a d i a (.££) , p r o t o z a (£J_) and m u l t i c e l l u l a r organisms such as s c h i s t o s m u l a o f Schistosoma mansoni . The p e r o x i d a s e i s a l s o t o x i c t o c e r t a i n m a m a l l i a n c e l l s , e.g. spermatozoa (£3.) , e r y t h r o c y t e s (2£) , l e u c o c y t e s (11) , p l a t e l e t s (12.) and tumor c e l l s (22.) i and i n a c t i v a t e s c e r t a i n s o l u b l e m e d i a t o r s such as t h e c h e m o t a c t i c f a c t o r , C5a (24.) • The p e r o x i d a s e can a l s o t r a n s f o r m 2

2

2

2

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Identifying Singlet Oxygen

4. KHAN

F i g u r e 5:

67

T

i

r

10

20

30

S t e r n - V o l m e r p l o t o f 1.28 m i c r o n e m i s s i o n o f s i n g l e t d e l t a m o l e c u l a r oxygen as a f u n c t i o n o f a s c o r b i c a c i d c o n c e n t r a t i o n . (Taken from Ref. 3 9 ) .

-0.50

* '

1200

i » '

I — i i i i l

1270

1340

WAVELENGTH (nm) Figure

6:

Near IR s i n g l e t oxygen e m i s s i o n p h o t o s e n s i t i z e d by d e m e c l o c y c l i n e (DMC), t e t r a c y c l i n e (TC), m i n o c y c l i n e (MC); oxygen s a t u r a t e d s o l v e n t [99.4% C C I 4 / O . 6 % Me2S0 ( v o l / v o l ) ] a t room t e m p e r a t u r e . (Taken from Ref. 5 4 ) .

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

68

LIGHT-ACTIVATED PESTICIDES

p r o s t a g l a n d i n s (25J , thus p o s s i b l y p l a y s a r e g u l a t o r y r o l e i n immune f u n c t i o n by m o d u l a t i n g t h e i n f l a m m a t o r y response. The mechanism o f a c t i o n o f t h e MPO system i s complex and c u r r e n t l y a c c e p t e d view i s as f o l l o w s (2£) :

H 0 2

2

+ CI" + H

+

-»

H0 2

+ H0C1

<->

H

+

+

the

0C1"

MPO R e c o g n i z i n g t h e c l a s s i c s i n g l e t oxygen g e n e r a t i n g r e a c t i o n hydrogen p e r o x i d e - h y p o c h l o r i t e ( 8 ) :

H

2°2

+

o c l

~

1

->0 ( ^g) 2

+ H0 2

of

+ CI"

i n t h e MPO enzyme mechanism t h e s i n g l e t oxygen g e n e r a t i n c h e m i c a l s c a v e n g e r s and d e u t e r i u m k i n e t i c e f f e c t s . They c o n c l u d e d t h a t c h e m i c a l e v i d e n c e s u p p o r t s s i n g l e t oxygen g e n e r a t i o n i n t h e MPO system ( 2 2 ) . U s i n g our G e - s p e c t r o m e t e r and w i t h t h e g i f t o f a sample o f m y e l o p e r o x i d a s e from D r s . Rosen and K l e b a n o f f , we have o b t a i n e d t h e c r i t i c a l s p e c t r a l e v i d e n c e o f s i n g l e t oxygen g e n e r a t i o n from t h e MPO/H 0 /Br" system ( F i g u r e 7 ) . We were a l s o a b l e t o e s t i m a t e t h e e f f i c i e n c y o f s i n g l e t oxygen g e n e r a t i o n i n t h i s system t o be about 0.5, i . e . two H 0 m o l e c u l e s y i e l d one m o l e c u l e o f 0 ( A g ) i n t h i s e n z y m a t i c system (2&). Kanofsky e t . a l . (79) have a l s o seen s i n g l e t oxygen g e n e r a t i o n i n t h e MPO system. They emphasize t h e n o n - p h y s i o l o g i c a l c o n d i t i o n s of the experiments. 2

2

1

f

2

2

2

L a c t o p e r o x i d a s e : M i l k and S a l i v a . L a c t o p e r o x i d a s e (LPO) i s s e c r e t e d i n t o s a l i v a by t h e human s a l i v a r y g l a n d s and i s a l s o p r o d u c e d by t h e mammary g l a n d s and found i n h i g h c o n c e n t r a t i o n i n milk, p a r t i c u l a r l y bovine milk. T h e o r e l l , e t a l . (80-81) were t h e f i r s t t o o b t a i n a h i g h l y p u r i f i e d p r e p a r t i o n o f LPO enzyme crystals. Klebanoff e s t a b l i s h e d the a n t i m i c r o b i a l a c t i v i t y of the L P O / H 0 / h a l i d e system (ILL) . We have o b t a i n e d s i n g l e t oxygen 2

2

e m i s s i o n from t h e LPO/H 0 /Br~ r e a c t i o n w i t h t h e Ge spectophotometer. Kanofsky (82) has p e r f o r m e d a k i n e t i c s t u d y o f t h e LPO r e a c t i o n . Our e s t i m a t e d e f f i c i e n c y o f s i n g l e t oxygen g e n e r a t i o n from t h e LPO r e a c t i o n i s comparable t o t h e e f f i c i e n c y o f t h e MPO r e a c t i o n , b e a r i n g out t h e i r s i m i l a r a n t i m i c r o b i a l a c t i o n (£2.) . MPO, however, o c c u r s i n s i d e t h e g r a n u l e s embedded i n t h e membrane o f t h e PMN and i s r e l e a s e d i n t o t h e phagosome on d e g r a n u l a t i o n by t h e a c t i v a t e d PMN, i n c o n t r a s t t o LPO which i s not confined i n vacules. 2

2

P l a n t Enzymes: C h l o r o p e r o x i d a s e . C h l o r o p e r o x i d a s e (CPO), was o r i g i n a l l y i s o l a t e d and c h a r a c t e r i z e d by M o r r i s and Hager ( M ) . CPO has an e f f e c t i v e c a t a l a s e - l i k e a c t i v i t y , as w e l l as e x h i b i t i n g t h e c l a s s i c a l p e r o x i d a t i v e and h a l o g e n a t i n g a c t i v i t y o f a p e r o x i d a s e (JL5.) . The enzyme can u t i l i z e b o t h c h l o r i d e and bromide i o n s f o r enzymic h a l o g e n a t i o n . Khan, Gebauer, and Hager examined t h e CPO/H 0 /C1~ enzyme system f o r s i n g l e t oxygen g e n e r a t i o n and 2

2

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

4. KHAN

69

Identifying Singlet Oxygen

o b t a i n e d a s t r o n g 1268 nm e m i s s i o n , shown i n F i g u r e 8 (J5_Q.) . T h i s i s t h e f i r s t r e p o r t e d spectrum o f s i n g l e t oxygen g e n e r a t e d i n an enzymic system. B i o s y n t h e t i c Enzymes: L i p o x y g e n a s e (86). The l i p o x y g e n a s e m e d i a t e d oxygen m o l e c u l e r e a c t i o n w i t h p o l y u n s a t u r a t e d f a t t y a c i d s y i e l d i n g h y d r o p e r o x i d e s i s o f fundamental i m p o r t a n c e i n l i p i d b i o c h e m i s t r y and i s t h e i n i t i a l s t e p i n t h e b i o s y n t h e s i s o f a h o s t of b i o l o g i c a l l y and m e d i c a l l y i m p o r t a n t m o l e c u l e s , t h e p r o s t a g l a n d i n s and l e u k o t r i e n e s (87-88). We o b s e r v e d a weak s i n g l e t oxygen e m i s s i o n from t h e l i p o x y g e n a s e r e a c t i o n w i t h e i t h e r l i n o l e n i c a c i d o r w i t h a r a c h i d o n i c a c i d as s u b s t r a t e . U s i n g t h e G e - s p e c t r o m e t e r , we s e a r c h e d f o r 12 68 nm e m i s s i o n o f s i n g l e t oxygen from L i p o x i d a s e , Type l / N a - l i n o l e a t e / 0 2 and L i p o x i d a s e , Type 1/Naa r a c h i d o n a t e / 0 2 r e a c t i o n s a t room t e m p e r a t u r e . A typical e x p e r i m e n t c o n s i s t s o f [ L i p o x i d a s e , Type 1 (Sigma C h e m i c a l C o . ) , 100 M-g/ml; N a - l i n o l e a t e (Sigma C h e m i c a l Co.), 40 mM; 0.1 M t r i s H C 1 , pH 9.2 b u f f e r w i t volume 15 m l ] , [20 s c a n s sec p e r nm, b a c k g r o u n d s u b t r a c t i o n ] y y (fl_2) , u s i n g a G e - k i n e t i c s p e c t r o m e t e r , o b s e r v e d t h e 12 68 nm s i n g l e t oxygen e m i s s i o n from t h e o x i d a t i o n o f l i n o l e i c a c i d c a t a l y z e d by soybean l i p o x y g e n a s e isozymes, m a i n l y from l i p o x y g e n a s e - 3 . From t h e i r i n v e s t i g a t i o n under o p t i m a l s i n g l e t oxygen g e n e r a t i n g c o n d i t i o n s , t h e y c o n c l u d e d t h a t a R u s s e l l l i k e mechanism (20.) of p e r o x y r a d i c a l r e c o m b i n a t i o n l e a d i n g t o s i n g l e t oxygen g e n e r a t i o n was q u i t e p l a u s i b l e . Thermal G e n e r a t i o n :

Dissociating

Endoperoxide

S i n g l e t oxygen, O2 (^Ag), r e a c t s w i t h p o l y c y c l i c h y d r o c a r b o n s t o produce e n d o p e r o x i d e s which, upon h e a t i n g , r e g e n e r a t e m o l e c u l a r oxygen and t h e p a r e n t h y d r o c a r b o n ( 1 0 . 9 1 - 9 3 ) . In t h e c a s e o f some of t h e t r a n s a n n u l a r p e r o x i d e s o f t h e n a p t h a l e n e and a n t h r a c e n e s e r i e s , c h e m i c a l r e a c t i v i t y s t u d i e s have shown t h a t a l a r g e f r a c t i o n , i f not a l l , o f t h e r e g e n e r a t e d oxygen appears t o be i n the s i n g l e t e x c i t e d s t a t e (11.94-95). W i l s o n , Khan, and M e h r o t r a (JL£) chose two e n d o p e r o x i d e s , 1.4-dimethyl-napthalene-l,4e n d o p e r o x i d e and 1,4-dimethoxy-9,10-diphenyl-anthraene-l,4endoperoxide t o s p e c t r a l l y i n v e s t i g a t e the g e n e r a t i o n of s i n g l e t oxygen i n t h e t h e r m a l d i s s o c i a t i o n o f t h e s e e n d o p e r o x i d e s . See F i g u r e 9. A l s o shown i n t h e f i g u r e i s t h e o b s e r v e d s p e c t r a l d i s t r i b u t i o n o f t h e t h e r m a l e m i s s i o n o f t h e s o l v e n t a t t h e same temperature. Note t h a t t h e t h e r m a l s p e c t r a l maximum d i s p l a y e d i s an a p p a r e n t one, not a t r u e maximum. Chou and F r e i (JLZ.) have r e p o r t e d t h e 1270 nm e m i s s i o n o f s i n g l e t oxygen from t h e t h e r m a l d i s s o c i a t i o n o f 1 , 4 - d i m e t h y l n a p t h a l e n e a t room t e m p e r a t u r e .

Chemical Generation:

Triethylsilane-Qzone Reaction

Corey, M e h r o t r a and Khan (j£ft) r e c e n t l y examined t h e r e a c t i o n o f t r i a l k y l s i l a n e w i t h ozone a t -75°C i n i n e r t o r g a n i c s o l v e n t s and f o u n d a h i g h l y e f f i c i e n t low-temperature s o u r c e f o r s i n g l e t d e l t a oxygen. U s i n g t h e G e - s p e c t r o m e t e r , t h e y c h a r a c t e r i z e d a f r e e l y d i f f u s i n g s i n g l e t d e l t a oxygen m o l e c u l e g e n e r a t e d from a r e a c t i o n intermediate. The i n t e r m e d i a t e i s t r i a l k y l s i l y l t r i o x i d e [ ( C 2 H 5 ) 3 S i O O O H ] w i t h an approximate h a l f l i f e o f 150 seconds i n methylene c h l o r i d e a t c_a. -60°C. Chemical t r a p p i n g experiments

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

LIGHT-ACTIVATED PESTICIDES

I.70E05

5.50 E03

I70E05

H 0 /NaOCI 2

2

3

(0,0) k g — Sg~

CO

z

-0.30E05 1200 1275 1350

-0.30E05 1200 12^5 1350

-0.50E03 ' 1200 1275 1350

WAVELENGTH, (nm)

F i g u r e 7:

Chemiluminescenc temperature i n t e n s i t y s c a l e i s p r o p o r t i o n a l to the number of photons e m i t t e d by the source ( n e g a t i v e numbers on the s c a l e a r e due to i n s t r u m e n t a l background subtraction). A. The 1268 nm e m i s s i o n o f s i n g l e t d e l t a d i o x y g e n from m y e l o p e r o x i d a s e r e a c t i n g w i t h H 2 O 2 i n t h e p r e s e n c e o f B r " . B. The 12 68 nm e m i s s i o n o f s i n g l e t d e l t a d i o x y g e n from t h e s t a n d a r d s i n g l e t oxygen g e n e r a t i n g r e a c t i o n 0 C 1 ~ • H 2 O 2 under comparable c o n d i t i o n s . C. The 12 68 nm e m i s s i o n from t h e 0 C 1 ~ * H 2 0 2 under n e a r optimum detection conditions.

(Adapted from R e f . 7 8 ) .

CHLOROPEROXIDASE

WAVELENGTH, MICRON F i g u r e 8:

N e a r - i n f r a r e d s i n g l e t oxygen c h e m i l u m i n e s c e n c e s p e c t r u m from t h e e n z y m a t i c r e a c t i o n o f chloroperoxidase with H 2 O 2 i n the presence of C l ~ . (Adapted from R e f . 58) .

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

4.

Identifying Singlet Oxygen

KHAN

71

1.01—i—r—j—i—|—i—i—i—r

>-

1100

1400

1700

WAVELENGTH (nm)

Figure

9:

1,4-dimethoxy-9,10-diphenylanthracene-l,4e n d o p e r o x i d e i n C C I 4 a t 50°C. A l s o shown a r e the s o l v e n t t h e r m a l e m i s s i o n g i v i n g an a p p a r e n t maximum at c_a, 1600 nm due t o a drop i n d e t e c t o r sensitivity. Two scans a r e shown, one t a k e n immediately a f t e r a d d i t i o n of the endoperoxide, the o t h e r a f t e r i t s complete d e c o m p o s i t i o n . (Adapted from R e f . 9 6 ) .

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

72

LIGHT-ACTIVATED PESTICIDES

estimated the e f f i c i e n c y of s i n g l e t The r e a c t i o n scheme i s as f o l l o w s : (C H ) SiH 2

5

3

+ 0

3

oxygen g e n e r a t i o n t o be 91%.

-> ( C H ) SiOOOH -> ( C H ) S i O H + 0 2

5

3

2

5

3

2

^Ag)

T h i s c o n v e n i e n t , h i g h l y e f f i c i e n t , low-temperature s i n g l e t oxygen s o u r c e may have wide a p p l i c a t i o n i n t h e s y n t h e s i s o f t h e r m a l l y l a b i l e s i n g l e t oxygen r e a c t i o n p r o d u c t s o f h y d r o p e r o x i d e s and endoperoxides. Conclusion. S i n g l e t oxygen r e s e a r c h r e p r e s e n t s a new c r o s s disciplinary effort. The m e t h o d o l o g i e s o f c h e m i c a l k i n e t i c s , p h o t o c h e m i s t r y , and p h o t o b i o l o g y c a n a l l be a p p l i e d t o t h e problem, s u b j e c t t o a s i n g l e r e s t r a i n t , t h a t t h e p r e s e n c e o f s i n g l e t oxygen i s unambiguously e s t a b l i s h e d . Spectroscopic techniques are the natural choice t o f u l f i l l t h i s task. In s p e c t r o s c o p y , however, d e t e c t i n g t h i s low o s c i l l a t o matrix i s d i f f i c u l t . Nea sensitive. The e m i s s i o g one o f t h e weakest known, s i n c e h i g h m e t a s t a b i l i t y makes t h i s s t a t e e x t r e m e l y s u s c e p t i b l e t o s o l v e n t q u e n c h i n g . The d e t e c t i o n l i m i t i n C C I 4 w i t h o u r i n s t r u m e n t i s about l O " ^ m o l e s / s e c . In H 0 s i n g l e t oxygen i s more d r a s t i c a l l y quenched and o n l y one photon i s e m i t t e d f o r e v e r y 10^ s i n g l e t oxygen m o l e c u l e s g e n e r a t e d , p u t t i n g t h e d e t e c t i o n l i m i t i n aqueous media a t 10"^ m o l e s / s e c . 1

2

Acknowledgments The a u t h o r acknowledges t h e generous h o s p i t a l i t y of P r o f e s s o r E . J . Corey. T h i s work was s u p p o r t e d b y t h e N a t i o n a l F o u n d a t i o n f o r Cancer Research, Bethesda, MD (Grant t o t h e I n s t i t u t e o f M o l e c u l a r B i o p h y s i c s , F l o r i d a S t a t e U n i v e r s i t y ) and by t h e N a t i o n a l S c i e n c e F o u n d a t i o n (Grant t o P r o f e s s o r E . J . Corey, Department o f C h e m i s t r y , H a r v a r d U n i v e r s i t y ) .

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2

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52. Hasan, T . ; Kochevar, I. E . ; McAuliffe, D. J.; Cooperman, B. S.; Abdulah, D. J . Invest. Dermatol. 1984, 83, 179-83. 53. Sandberg, S. O.; Glette, J.; Hopen, G.; Solberg, C. O. Photochem. Photobiol. 1984, 39, 43-48. 54. Hasan, T . ; Khan, A. U. Proc. Natl. Acad. Sci USA 1986, 83, 4604-06. 55. Krishnamurty, H. G.; Simpson,F. J . J . Biol. Chem. 1970, 245, 1467-71. 56. Matsuura, T.; Matsushima, H . ; Sakamoto, H. J. Am. Chem. Soc., 1967 89, 6370-71. 57. Allen, R. C.; Stjernholm, R. L.; Steele, R. H. Biochem. Biophys. Res. Commun. 1972, 47, 679-84. 58. Khan, A.U.; Gebauer, P.; Hager, L. P. Proc. Natl. Acad. Sci. USA 1983, 80, 5195-97. 59. Kanofsky, J . R. J . Biol. Chem. 1984, 259, 5996-00. 60. Agner, K. In Structure and Function of Oxidation-Reduction Enzymes: Akeson, A.; Ehrenberg, A., Eds.; Pergamon, Oxford, 1972; pp 329-35. 61. Klebanoff, S. J . J 62. Klebanoff, S. J . J 63. Diamond, R. D.; Clark, R. A.; Haudenschild, C. C. J . Clin. Inyjast. 1980, 66 908-17. 64. Belding, M. E.; Klebanoff, S. J.; Ray, C. G. Science. 1970, 167, 195-96. 65. Jacobs, A. A.; Low, I. E . ; Paul, B. B.; Strauss, R. R.; Sbarra, A. J . Infect. Immun. 1972, 2, 127-31. 66. Yong, E. C.; Kno, C. C.; Klebanoff, S. J . Am. Soc. Microbiol. Abst. Annual Meeting 1980, p. 38. 67. Chang, K. -P. J . Trop. Med. Hyg. 1981, 30, 322-33. 68. Jong, E. C.; Mahmoud, A. A. F . ; Klebanoff, S.J. J . Immunol. 1981, 126, 468-71. 69. Klebanoff, S. J.; Smith, D. C. Biol. Reprod. 1970, 3, 236-42. 70. Klebanoff, S. J.; Clark, R. A. Blood 1975, 45, 699-07. 71. Clark, R. A.; Klebanoff, S. J . Blood 1977, 50, 65-70. 72. Clark, R. A.; Klebanoff, S. J . J . Clin. Invest. 1979, 63 17783. 73. Clark, R. A.; Klebanoff, S. J.; Einstein, A. B. Blood 1975, 45, 161-70. 74. Clark, R. A.; Klebanoff, S. J . J . Clin. Invest. 1979, 64 91320. 75. Paredes, J.; Weiss, S. J . J . Biol. Chem. 1982, 257 2738-40. 76. Klebanoff, S. J . In Phagocytic Cells; Gallin, J . I.; Fauci, A. S.; Eds.; Raven Press, New York, 1982; pp 111-62. 77. Rosen, H . ; Klebanoff, S.J. J . Biol. Chem. 1977, 252, 4803-10. 78. Khan, A. U. Biochem. Biophys. Res. Commun. 1984, 122. 668-75. 79. Kanofsky, J . R.; Wright, J.; Miles-Richardson, G. E.; Tauber, A. I. J . Clin. Invest. 1984, 74 1489-95. 80. Theorell, H.; Akeson, A. Arkiv. Kemi. Mineral. Geol. 1943, 17B, No. 7. 81. Theorell, H.; Paul, K. G. Arkiv. Kemi, Mineral. Geol. 1944, 18A, No. 12. 82. Kanofsky, J . R. J . Biol. Chem. 1983, 258, 5991-93. 83. Khan, A. U . , J . Am. Chem. Soc. 1983, 105, 7195-97. 84. Morris, D. R.; Hager, L. P. J . Biol. Chem. 1966, 241, 1763-68. 85. Thomas, J . A.; Morris, D. R.; Hager, L. P. J . Biol. Chem. 1970, 2A2, 3129-34. 86. Khan, A. U. unpublished. 87. Samuelson, B. Science 1983, 220, 568-75. 88. Corey, E. J. Experientia 1982, 38, 1259-1381.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

4. KHAN

Identifying Singlet Oxygen

75

89. Kanofsky, J . R.; Axelrod, B. J . Biol. Chem. 1986, 261, 1099104. 90. Russell, G. A. J . Am. Chem. Soc. 1957, 79, 3871-77. 91. Moureu, C.; Dufraisse, C.; Dean, P. M. C.R. Acad. Sci. 1926, 198, 1584-85. 92. Dufraisse, C . ; Velluz, L . ; Velluz, L. C.R. Acad. Sci. 1939, 208, 1822-24. 93. Rigaudy, J.; Guillaume, J.; Maurette, D. Bull. Soc. Chem. Fr. 1971, 144-52. 94. Wasserman, H. H.; Scheffer, J. R.; Cooper, J. L. J. Am. Chem. Soc. 1972, 94, 4991-96. 95. Wilson, T. Photochem. Photobiol. 1969, 10, 441-44. 96. Wilson, T . ; Khan, A. U.; Mehrotra, M. M. Photochem. Photobiol. 1986, 43, 661-62. 97. Chou, P. T . ; Frei, H. Chem. Phys. Lett. 1985, 122, 87-91. 98. Corey, E. J.; Mehrotra, M. M.; Khan, A. U. J . Am. Chem. Soc. 1986, 108, 2472-73. RECEIVED February 11, 198

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 5

Singlet Oxygen Quantum Yields Michael A. J. Rodgers Center for Fast Kinetics Research, University of Texas at Austin, Austin, TX 78712

The lowest excited stat gen) has the spectroscopi level lies 7880 cm (1eV; 23kcal/mol) above the v=0 level of the molecular ground state (3Σ-g) The Δ —> 3Σ-g transition and its inverse are strongly forbidden for electric dipole radiation in the isolated molecules -- at zero pressure in the gas phase the radiat ive lifetime of O ( Δ ) is calculated to be 45 mins.(1). This property is apparently phase dependent since a value of 4s has been reported in carbon tetrachloride (2). This forbiddeness of the op tical transition makes generation of O ( Δ ) by direct-photon ab sorption very difficult to accomplish although quantities sufficient to allow kinetic studies in Freons have been produced by irradiation of high pressures of O in Freon solution with 1.064 μm radiation from a high power Nd: YAG laser (3). The extremely low probability of the radiative transition has several consequences, the one that is pertinent to this account concerns the use of indirect methods of producing O ( Δ ) for quan titative kinetic studies. Such investigations are generally per formed in one of two ways. (i) Singlet oxygen is formed at a constant rate by applica tion of some perturbation that operates continuously. The progress of reactions are followed by measuring the yields of chemical prod ucts or other effects as a function of time over which the pertur bation is continued. (ii) Singlet oxygen is formed by a short high, intensity burst of the perturbing effect such that the concentration of singlet oxygen produced is sufficient to be followed, either directly or indirectly, in timer-resolved experiments. Both kinds of experiment are capable of yielding kinetic data of interest such as natural lifetimes, reaction rate constants, quenching rate constants and so forth. The perturbation effect most often employed is that of photo excitation of a sensitizer. This act forms upper singlet sensi tizer states that can undergo inter-system crossing to triplet states (Reactions 1-4). -1

1

g

1

2

g

1

2

g

2

1

2

g

0097-6156/87/0339-0076$06.50/0 © 1987 American Chemical Society

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

5.

RODGERS

Singlet Oxygen Quantum Yields S + hv 1 *

> „

S

1

S* 1 * S

Subsequently, (5) and (6)

77 (1)

1s*

>

s

> >

s

+

h

V

( )

F

2

3s*

(3) (4)

the t r i p l e t s t a t e can decay a c c o r d i n g t o R e a c t i o n s

3s* 3 *

> o

~

S

S + hv s

(5) (6)

p

In the above scheme hvp and hvp r e f e r t o the r a d i a t i v e p r o c e s s e s denoted as f l u o r e s c e n c e ( R e a c t i o n 2) and phosphorescence ( R e a c t i o n 5) r e s p e c t i v e l y . For most s e n s i t i z e r s i n f l u i d media t h e phosphorr e s c e n t channel c o n t r i b u t e s o n l y m i n i m a l l y t o the t o t a l decay o f the t r i p l e t s t a t e p o p u l a t i o n . When oxygen i s d i s s o l v e n o r m a l l y employed) the f o l l o w i n g a d d i t i o n a l r e a c t i o n s a r e p o s s i b l e . is*

+

0 (3z )

-

1s*

• 0 (3zg)

-

1s*

+

°2( ^i)

~

3s*

• 0 (3z-g)

-

3S*

+

-

2

g

2

3

2

0 (3z-g) 2

> > > > >

3s*

+ 0 (lA ) 2

(7)

g

(8)

1

s + 0 ( A ) 2

g

s + 0 (3z )

(9)

s + 0 (3z )

(10)

s + 0 (lA )

(11)

2

2

2

g

g

g

C l e a r l y R e a c t i o n (7) i s s p i n - a l l o w e d but i s o n l y p o s s i b l e when (E -ET^EA). R e a c t i o n (8) i s s p i n f o r b i d d e n and R e a c t i o n (9) has s e v e r e Franck.-Condon r e s t r i c t i o n s i n t h a t the energy o f 1s* has t o be d i s s i p a t e d i n t o v i b r a t i o n a l modes. S i m i l a r r e s t r i c t i o n s a p p l y to R e a c t i o n (10) which i s i n c o m p e t i t i o n w i t h R e a c t i o n (11), t h e s i n g l e t oxygens-producing channel from s e n s i t i z e r t r i p l e t s t a t e s . S

RATIONALE FOR MEASURING SINGLET OXYGEN QUANTUM YIELDS The r e a s o n why t h e r e s h o u l d be s o much i n t e r e s t i n d e t e r m i n i n g quantum y i e l d s o f s i n g l e t oxygen f a l l i n t o two major c a t e g o r i e s , one c o n c e r n i n g fundamental p h o t o p h y s i c s , the o t h e r c o n c e r n i n g apr plications of photosensitized oxidation. The p h o t o p h y s i c s requirement concerns expanding our knowledge about the i n t e r a c t i o n s o f the s e n s i t i z e r e x c i t e d s t a t e s w i t h oxygen as summarized i n R e a c t i o n s (7) through (11) above. Q u a n t i t a t i v e measurement o f the y i e l d s of 0 2 ( A ) produced from m o l e c u l a r singr. l e t s t a t e s and m o l e c u l a r t r i p l e t s t a t e s a i d s i n a s s e s s i n g the amount t h a t a p a r t i c u l a r r e a c t i o n c c h a n n e l c o n t r i b u t e s t o the over* a l l d e a c t i v a t i o n . I n f o r m a t i o n on how the y i e l d s v a r y w i t h i n f l u r ences such as s e n s i t i z e r s t r u c t u r e , s t a t e energy, n a t u r e o f s o l v e n t and s o on i s i m p o r t a n t i n p r o v i d i n g m e c h a n i s t i c i n f o r m a t i o n . The quenching o f e x c i t e d s t a t e s by oxygen i s such a w e l l know p r o c e s s t h a t i t may be s u r p r i s i n g t o many t o l e a r n t h a t i t i s s o l i t t l e understood. 1

g

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

78

LIGHT-ACTIVATED PESTICIDES

The a r e a o f p h o t o s e n s i t i z e d o x i d a t i o n s i s v e r y wide, r a n g i n g over a l l systems where the combined a c t i o n of v i s i b l e l i g h t , an a b s o r b i n g m o l e c u l e or r e s i d u e , and m o l e c u l a r oxygen can r e s u l t i n p h o t o c h e m i c a l damages ( 5 ) . T h i s c o m b i n a t i o n has impact i n such d i v e r s e p l a c e s as the t e x t i l e i n d u s t r y , the cancer c l i n i c and the p l a n t l e a f . T e x t i l e t e c h n o l o g i s t s a r e concerned w i t h the photof a d i n g and p h o t o d e s t r u c t i o n o f f i b e r s t h a t have been dyed w i t h v i s i b l e l i g h t a b s o r b i n g pigments; i n the cancer c l i n i c t r i a l s a r e p r o c e e d i n g i n which p o r p h y r i n doped tumors undergo n e c r o s i s when i r r a d i a t e d with red l i g h t ; the p h o t o s y n t h e t i c a p p a r a t u s o f green p l a n t s a r e i d e a l l y s u i t e d f o r p r o d u c i n g s i n g l e t oxygen from photoe x c i t e d c h l o r o p h y l l m o l e c u l e s -.- the presence o f c a r o t e n o i d s o f f e r s non-damaging p h y s i c a l modes o f d e a c t i v a t i n g c h l o r o p h y l l t r i p l e t s t a t e s . P r o c e s s e s such as t h e s e , t o g e t h e r w i t h the a c t i o n o f l i g h t - a c t i v a t e d p e s t i c i d e s , f a l l under the g e n e r a l heading of photodynamic a c t i o n , i e damage i n c u r r e d i n a b i o l o g i c a l system through the p h o t o s e n s i t i z e d o x i d a t i o e v i d e n c e i n d i c a t i n g th r e a c t i v e s p e c i e s i n on photodynami more q u a n t i t a t i v e i n f o r m a t i o n t h a t we can o b t a i n about the y i e l d s of s i n g l e t oxygen i n photodynamic c i r c u m s t a n c e s , then the g r e a t e r our o p p o r t u n i t y f o r u n d e r s t a n d i n g the d e t a i l e d mechanism and a l t e r r i n g , (enhancing or d i m i n i s h i n g , a c c o r d i n g t o the r e q u i r e m e n t s ) i t s effects. SINGLET STATE SOURCES A c o n s i d e r a t i o n o f R e a c t i o n s ( 7 ) , (10) and (11) shows t h a t f o r s e n s i t i z e r s h a v i n g a s u f f i c i e n t S-T energy d i f f e r e n c e , each ^S* s t a t e (_ie each photon) w i l l g i v e r i s e , i n the l i m i t , t o two 0 2 ( ^ A ) m o l e c u l e s — R e a c t i o n (7) f o l l o w e d by R e a c t i o n ( 1 1 ) , _ie t h e s i n g l e t oxygen quantum y i e l d (*^) can approach 2.0. Several researchers (7-9) have i n v e s t i g a t e d such p r o c e s s e s and e v i d e n c e f o r *^ v a l u e s g r e a t e r than u n i t y has been o b t a i n e d . S u b s t i t u t e d anthracenes and h i g h e r homologues show t h i s e f f e c t . Of c o u r s e , the l i m i t i n g quanc-turn y i e l d i s r a r e l y a c h i e v e d s i m p l y because a t oxygen c o n c e n t r a tions attainable i n 0 -saturated organic solvents ( t y p i c a l l y l O ^ M ) , the product ky[02] i s u s u a l l y unable t o outweigh the sum ( k + k3 + kij) i e , s i n g l e t s t a t e s a r e g e n e r a l l y l o s t t o the unimol e c u l a r decay modes w i t h a p p r o x i m a t e l y s i m i l a r e f f i c i e n c y t o t h a t w i t h which they a r e quenched by oxygen. g

2

2

TRIPLET STATE SOURCES With m o l e c u l e s t h a t have Es - Ej< E^, R e a c t i o n (7) i s not energetic a l l y f e a s i b l e and s i n c e R e a c t i o n (8) i s s p i n - f o r b i d d e n , the o n l y source o f 0 ( A ) from e x c i t e d s t a t e s o f many systems i s through the t r i p l e t m a n i f o l d s v i a oxygen quenching. I n many m o l e c u l a r systems, oxygen quenching o f t r i p l e t s t a t e s i s the o n l y p r o c e s s f o r s i n g l e t oxygen p r o d u c t i o n . R e a c t i o n (11) above can be expanded i n t o the s e t ( R e a c t i o n s 11a-c) as below and i n q u a n t i t a t i v e terms, c o m p e t i t i o n between the s e t determines the r a t e s o f 0 ( A ) f o r m a t i o n and t h e quantum yields. 1

2

g

1

2

g

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

5.

RODGERS

Singlet Oxygen Quantum Yields

3s* + 02(3zg) ^==i

79

1(S—0 )»

->

S + 0 ( A )

(11a)

3(S—0 )*

->

S + 0 (3Eg)

(11b)

2

2

1

2

G

2

5(S~0 )*

(11c)

2

The s p e c i e s 1 i 3 , 5 ( s — 0 ) * r e p r e s e n t s the t r a n s i t i o n s t a t e between r e a c t a n t s and p r o d u c t s . The mutual t r i p l e t m u l t i p l i c i t y of the r e a c t a n t s conveys n i n e p o s s i b l e degenerate s p i n s u b ^ s t a t e s w i t h i n the t r a n s i t i o n s t a t e ; the s i n g l e t and t r i p l e t e n t i t i e s have e n e r g e t i c a l l y r e a c h a b l e product s t a t e s but the q u i n t e t s have not and they must r e t u r n t o r e a c t a n t s t a t e s . The consequence o f t h i s i s t h a t the r a t e c o n s t a n t f o r s i n g l e t oxygen f o r m a t i o n w i l l not be l a r g e r than one n i n t h of the v a l u e of the r a t e c o n s t a n t f o r d i f f u s i o n - l i m i t e d quenching i n the medium (J_0). That the measured r a t e c o n s t a n t s f o r oxygen quenching o f the t r i p l e t s t a t e s o f many d i f f u s i o n c o n t r o l valu an i n d i c a t i o n (1_0) t h a single ( R e a c t i o n 11a) p r o v i d e s the o n l y t r i p l e t d e a c t i v a t i o n mechanism, t h e r e b y l e a d i n g t o 0 ( A ) quantum y i e l d s o f u n i t y . However, more d e t a i l e d o b s e r v a t i o n s show (11-13) t h i s i s not n e c e s s a r i l y t r u e and t h a t the system i s more c o m p l i c a t e d than f i r s t thought. The y i e l d o f 0 ( l A ) formed from a m o l e c u l a r t r i p l e t s t a t e i n v o l v e s a c o m p e t i t i o n between R e a c t i o n s (10) and (11) and the parameter S was c o i n e d (1J_) t o d e s c r i b e t h i s where 2

1

2

2

g

g

A

SA = k i / ( k 1

1 1

+

k ) 1 0

or, S i s the f r a c t i o n o f t r i p l e t quenchings by oxygen t h a t l e a d s t o s i n g l e t oxygen. Thus i n a p h o t o c h e m i c a l system t h a t i n v o l v e s 0 ( ^ A g ) f o r m a t i o n v i a s e n s i t i z e r t r i p l e t s t a t e s o n l y , we see t h a t A

2

S

*A = A'*T where <&T i s the t r i p l e t quantum y i e l d and $A i s the o v e r a l l s i n g l e t oxygen quantum y i e l d . T h e r e f o r e , a q u a n t i t a t i v e d i s c u s s i o n of s i n g l e t oxygen quantum y i e l d s must always take i n t o account the involvement of the independent v a r i a b l e s and *T. SINGLET OXYGEN YIELD MEASUREMENTS Measurements of s i n g l e t oxygen have been c a r r i e d out u s i n g b o t h s t e a d y - s t a t e and p u l s e d i l l u m i n a t i o n methods, but i t has never proved an easy m o l e c u l e t o measure l a r g e l y because i t i s not amenable to o p t i c a l absorption spectroscopy using conventional i n s t r u ments. E a r l i e r means of d e t e c t i o n employed c h e m i c a l t r a p s , i e , molecules that react with 0 ( l A ) r e s u l t i n g i n l o s s of s t a r t i n g m a t e r i a l (M) and/or p r o d u c t i o n of a r e l a t i v e l y s t a b l e peroxy produ c t , o r a t h e r m a l l y - a c t i v a t e d decay product t h e r e o f . 2

M + 02(1 A ) g

>

M0

g

2

> product

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

(13)

80

LIGHT-ACTIVATED PESTICIDES

Perhaps t h e most w i d e l y - u s e d s u b s t r a t e (M) has been d i p h e n y l i s o b e n z o f u r a n (DPBF) which, on r e a c t i o n w i t h s i n g l e t oxygen l o s e s i t s c h a r a c t e r i s t i c y e l l o w c o l o r . I n i t i a l l y DPBF was employed (14-17) i n s i t u a t i o n s where k i n e t i c d a t a were b e i n g e v a l u a t e d . L a t e r t h e e x t e n t o f b l e a c h i n g became employed i n y i e l d e v a l u a t i o n s (11,18-20). Singh e t a l (21_) have o u t l i n e d t h e p o t e n t i a l d i f f i c u l t t i e s i n u s i n g DPBF as a q u a n t i t a t i v e probe f o r s i n g l e t oxygen i n c o n t i n u o u s i l l u m i n a t i o n c o n d i t i o n s . I n t i m e r - r e s o l v e d experiments these problems a r e l e s s s e v e r e but c a u t i o n i s always n e c e s s a r y on account o f the m o l e c u l e ' s h i g h p h o t o s e n s i t i v i t y . L i k e DBPF, some condensed p o l y c y c l i c a r o m a t i c hydrocarbons ( r u b r e n e , 9,10 d i p h e n y l anthracene) form endoperoxides w i t h 0 2 ( A ) - a r e a c t i o n t h a t r e moves extended chromophores and l e a d s t o b l e a c h i n g . These m o l e c u l e s have a l s o been used f o r q u a n t i t a t i v e y i e l d e v a l u a t i o n s (7-9,22,23) In d e t e r m i n i n g t h e y i e l d o f s i n g l e t oxygen from a s e r i e s o f r o s e bengal d e r i v a t i v e s n y l - d i o x e n e as a probe a c y c l i c e s t e r which was q u a n t i f i e d gas c h r o m a t o g r a p h i c a l l y (24,25). Another method has been t o use p a r a - n i t r o s o d i m e t h y l a n i l i n e (RNO) a g r e e n - c o l o r e d m o l e c u l e - as a r e a c t i v e probe f o r t r a n s a n n u l a r p e r o x i d e s (M0 ) formed from s i n g l e t oxygen r e a c t i o n w i t h i m i d a z o l e s . A g a i n t h e c o l o r a t i o n o f M i s l o s t and t h i s p r o p e r t y i s f o l lowed q u a n t i t a t i v e l y t o r e s u l t i n * v a l u e s (23,26). Those c h e m i c a l probe systems t h a t depend on a s i m p l e c o l o r change can be, and have been employed i n both s t e a d y - s t a t e and t i m e - r e s o l v e d e x p e r i m e n t a l t i o n . Of c o u r s e i n u s i n g r e a c t i v e monitor m o l e c u l e s (DPBF, rub«r e n e , e t c . ) f o r quantum y i e l d measurement i t i s i m p o r t a n t t o know what f r a c t i o n o f t h e d e a c t i v a t i n g encounters l e a d s t o s u b s t r a t e l o s s . For DPBF t h i s f r a c t i o n i s a p p a r e n t l y u n i t y (27). I n r e c e n t y e a r s r e s e a r c h e r s have been u s i n g such i n d i r e c t c h e m i c a l probe t e c h n i q u e s l e s s , and newly-developed d i r e c t spectror. s c o p i c methods more. The use o f c h e m i c a l probes can be s u b j e c t t o problems a r i s i n g o u t o f u n c e r t a i n t i e s i n r e a c t i o n mechanisms w i t h d i f f e r e n t s e n s i t i z e r s . A l s o they r e q u i r e extreme c a r e i n e x c l u s i o n o f extraneous l i g h t . The advent o f f a s t response d e t e c t o r s w i t h i n f r a r e d s e n s i t i v i t y and h i g h bandwidth, h i g h g a i n a m p l i f i e r s has g i v e n t h e c a p a b i l i t y o f d e t e c t i n g t h e very weak luminescence a t 1.269 urn ( F i g u r e 1) r e s u l t i n g from the 3z~ < l A transition i n oxygen. The f o r b i d d e n e s s o f t h i s t r a n s i t i o n r e s u l t s i n l u m i n e s r cence quantum y i e l d s b e i n g v e r y low. The e a r l i e s t work i n t h i s a r e a was c a r r i e d o u t by R u s s i a n workers who p i o n e e r e d luminescence d e t e c t i o n i n both c.w. (28) and time«-.resolved modes ( 2 9 , 3 0 ) . These e a r l y e f f o r t s were r a p i d l y f o l l o w e d by a c t i v i t y i n t h e U.S. where r e d - s e n s i t i v e p h o t o m u l t i p l i e r d e t e c t o r s were r e p l a c e d by i n f r a r e d d e t e c t i n g photodiodes backed-up by h i g h g a i n a m p l i f i e r s f o r cont i n u o u s wave (c.w.) ( 3 D and t i m e - r e s o l v e d work (32-35). This t e c h n o l o g y has r e v o l u t i o n i z e d 0 2 ( A ) d e t e c t i o n because o f i t s d i r e c t n e s s , p r e c i s i o n , convenience and r a p i d i t y . The v a s t m a j o r i t y o f r e s e a r c h on 0 ( A ) u s i n g i n f r a r e d luminescence t e c h n i q u e s has concerned k i n e t i c s t u d i e s but t h e s e methods a r e a l s o a p p l i c a b l e t o quantum y i e l d s t u d i e s once t h e proper c a l i b r a t i o n has been c a r r i e d o u t . T h i s c a l i b r a t i o n p r o c e s s i s e s s e n t i a l and i t i s d e s c r i b e d i n some d e t a i l below. 1

g

2

A

g

1

g

1

2

g

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

RODGERS

1200

Singlet Oxygen Quantum Yields

nm

1300

F i g u r e 1. Luminescence from s i n g l e t oxygen i n a e r a t e d benzene solution. The s e n s i t i z e r was 2-acetonaphthone e x c i t e d a t 365 nm. The bandwidth a t FWHM i s 20 nm. Taken w i t h t h e a p p a r a t u s (Rodgers, M.A.J., t o be p u b l i s h e d ) shown i n f i g u r e 2.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

82

LIGHT-ACTIVATED PESTICIDES

Another p h y s i c a l t e c h n i q u e t h a t has r e c e n t l y been developed i s the use of t h e r m a l l e n s i n g . T h i s depends upon the d e a c t i v a t i o n of a p o p u l a t i o n o f e x c i t e d m o l e c u l e s by non.-radiative modes, _ i e , the e x c i t a t i o n energy i s r e l e a s e d t o the s u r r o u n d i n g medium as t h e r m a l energy. T h i s heat r e l e a s e , i f r a p i d enough, i e a f t e r l a s e r p u l s e e x c i t a t i o n , produces l o c a l changes i n t e m p e r a t u r e , d e n s i t y and r e f r a c t i v e index i n the medium. Thus the sample behaves momentari l y as a d i v e r g i n g l e n s and a c a r e f u l l y a l i g n e d o p t i c a l system can be s e t up t o probe the t r a n s i e n t l e n s and the r e s u l t i n g s i g n a l c o n t a i n s i n f o r m a t i o n on b o t h the k i n e t i c s of the n o n ^ r a d i a t i v e decay c h a n n e l s and on r e l a t i v e magnitudes of the c o n t r i b u t i o n s from the v a r i o u s decay modes. S i n c e s i n g l e t oxygen decays almost e x c l u s i v e l y n o n - r a d i a t i v e l y , i t i s p a r t i c u l a r l y w e l l ^ s u i t e d f o r thermal l e n s i n g s t u d i e s . Fuke, e t a l (36) f i r s t used t h i s t e c h n i q u e f o r k i n e t i c measurements and i t has r e c e n t l y been r e f i n e d and q u a n t i f i e d f o r y i e l d measurements by R o s s b r o i c h et a l (37). The method o f f e r s the c a p a b i l i t y o f measurin * d $T f o p h o t o e x c i t e d mole c u l e s by the r e l a t i v e l slow heat c o n t r i b u t i o n tively (37). APPARATUS FOR

INFRARED LUMINESCENCE MEASUREMENT

F u l l e r d e s c r i p t i o n s of the i n f r a r e d d e t e c t i o n methodology o c c u r elsewhere i n t h i s volume (3j3). B r i e f l y p r e s e n t e d here a r e two systems e x t a n t i n the a u t h o r s l a b o r a t o r y f o r c.w. and t i m e - r e s o l v e d quantum y i e l d measurements. 1

C.W.

EXCITATION

T h i s i s shown s c h e m a t i c a l l y i n F i g u r e 2 and i s a development o f systems used by Khan ( 3 9 ) , Kanofsky, (40) and H a l l and C h i g n e l l (Photochem. P h o t o b i o l . , i n p r e s s ) . S o l u t i o n s i n the 10mm x 10mm sample c u v e t t e are i r r a d i a t e d w i t h l i g h t from a 100W Hg a r c f i l r t e r e d through 10 cm of water and a heat a b s o r b i n g f i l t e r ( S c h o t t KG-3). T h i s c o m b i n a t i o n t r a n s m i t s mercury l i n e s a t 365 nm and above w i t h ca 90% e f f i c i e n c y but s t o p s r a d i a t i o n above 1000 nm w i t h h i g h e f f i c i e n c y . Luminescence i s c o l l e c t e d a t r i g h t a n g l e s by an Anaspec C a s s e g r a i n m i r r o r system ( f / 1 . 0 ) which conveys the l i g h t t o a monochromator ( O r i e l ) w i t h a 600 l i n e s per mm, 1.0 ym b l a z e g r a t ing. Monochromated r a d i a t i o n from the o u t p u t s l i t i s f o c u s s e d o n t o a 5mm germanium c r y s t a l PN d e t e c t o r c o u p l e d t o a transimpedance p r e a m p l i f i e r ( N o r t h Coast O p t i c a l Systems and S e n s o r s ) . B o t h det e c t o r s and p r e a m p l i f i e r a r e c o o l e d t o 77 K. T h i s system has a r e s p o n s i v i t y o f 5 x 109 v/W. THe d e t e c t o r i s covered by a 5 mm t h i c k d i s c of AR-coated h i g h p u r i t y s i l i c o n metal a c t i n g as an 1100 nm c u t - o f f f i l t e r . The e x c i t a t i o n beam i s chopped a t 100 Hz and the o u t p u t from the p r e a m p l i f i e r i s f e d t o a l o c k - i n a m p l i f i e r ( P r i n c e t o n A p p l i e d Research 124A) i n c o r p o r a t i n g a m u l t i r a n g e v o l t meter. A spectrum o f s i n g l e t oxygen measured w i t h t h i s i n s t r u m e n t i s shown i n F i g u r e 1. 2

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

RODGERS

Singlet Oxygen Quantum Yields

F i g u r e 2. I n f r a r e d emission spectrophotometer schematic. L: 100W Hg a r c w i t h l e n s assembly; S: s h u t t e r ; Ch: v a r i a b l e f r e q u e n c y chopper; WF: 10 cm water f i l t e r ; F<| : lamp f i l t e r s in»c l u d i n g KG3 heat f i l t e r and Hg l i n e f i l t e r ; C: 10 mm sample c u v e t t e ; SM; s p h e r i c a l m i r r o r ; CC: C a s s e g r a i n f/1 l i g h t c o l l e c r t o r ; F c u t o f f and o r d e r b l o c k i n g f i l t e r s ; M: monochromator; D: Germanium d e t e c t o r and a m p l i f i e r ; PSD: phase s e n s i t i v e detector. 2

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

84

LIGHT-ACTIVATED PESTICIDES

PULSED EXCITATION

The c u r r e n t v e r s i o n of t h i s apparatus ( F i g u r e 3a) v a r i e s o n l y marg i n a l l y from t h a t d e s c r i b e d e a r l i e r (35,41). The e x c i t a t i o n beam i s a 10 ns p u l s e from Qc-.switched Nd:YAG l a s e r o p e r a t i n g i n c o n j u c t i o n w i t h harmonic g e n e r a t o r s t o produce l i g h t a t 532 nm or 355 nm. A t t e n u a t i o n i s arranged by p l a c i n g a q u a r t z d i v e r g i n g l e n s ( c 5 mm) about 12 i n c h e s from the c u v e t t e p o s i t i o n and i n t e r p o s i n g n e u t r a l d e n s i t y f i l t e r s f o r f u r t h e r decreases. The 10 mm x 10 mm sample c u v e t t e i s h e l d c l o s e l y a g a i n s t a d i s c o f AR-coated s i l i c o n m e t a l ( a t 90 degrees t o the i n p u t f a c e ) immediately behind which i s p l a c e d a r e v e r s e - b i a s e d germanium photodiode (Judson J16) o p e r a t i n g photor-.conductively. The p h o t o d i o d e o u t p u t i s t a k e n t o a p r e a m p l i f i e r (Judson) w i t h a 500fl output impedance. A second a m p l i f i e r w i t h 10KP. i n p u t impedance and 50fl output impedance p r o v i d e s a f u r t h e r g a i n o f 10 and impedance matching t o the 508 i n p u t of a Bion mation 8100 waveform d i g i t i z e r a m p l i f i e r has been m o d i f i e range 50ft t o 1Kfl. The c u v e t t e , d e t e c t o r and p r e a m p l i f i e r system are c o n t a i n e d i n an aluminum e n c l o s u r e w i t h minimumrsized e n t r a n c e and e x i t h o l e s f o r the l a s e r beam. T h i s p r e v e n t s RF i n t e r f e r e n c e from the l a s e r lamps and Q-switch. The a p p a r a t u s has been conn s t r u c t e d such t h a t p h o t o d e t e c t o r s of d i f f e r e n t s i z e s may be used. S m a l l e r a r e a d e t e c t o r s a l l o w improvements i n s i g n a l r i s e t i m e , up t o a l i m i t s e t by the p r e a m p l i f i e r a n a l o g bandwidth (15 MHz). Rise times of 60 ns have been o b t a i n e d w i t h t h i s apparatus. F i g u r e 3b shows some t y p i c a l data o b t a i n e d w i t h t h i s d e v i c e . QUANTUM YIELD MEASUREMENTS USING INFRARED LUMINESCENCE Measurement methods based on l i g h t a b s o r p t i o n a r e r e l a t i v e and t h e r e f o r e have i n - b u i l t c a l i b r a t i o n s . T h i s i s not so f o r emisr. s i o n - m e a s u r i n g systems w h i c h measure photons a b s o l u t e l y and t h u s , i n q u a n t i t a t i v e y i e l d measurements, account has t o be t a k e n o f f a c t o r s such as l i g h t c o l l e c t i o n geometry, monochromator throughput e f f i c i e n c y and the d e t e c t o r response w i t h w a v e l e n g t h . In s h o r t , the i n s t r u m e n t must be c a l i b r a t e d . T h i s can most e x p e d i t i o u s l y be a c c o m p l i s h e d by employing r e f e r e n c e systems of known l u m i n e s c e n t quantum y i e l d . For s i n g l e t oxygen measurements t h i s r e f e r e n c e may have been o b t a i n e d e i t h e r t h r o u g h p h o t o c h e m i c a l y i e l d measurements o r from i n f r a r e d l u m i n e s c e n t measurements. Our approach has been a combination o f the two. The a b s o l u t e s t a n d a r d t h a t we developed a r i s e s out of measure.ments of the s i n g l e t oxygen luminescence s i g n a l (1^) immediately a f t e r a l a s e r p u l s e measured as a f u n c t i o n o f l a s e r energy (Gorman, A.A. e t a l , s u b m i t t e d f o r p u b l i c a t i o n ) The c h e m i c a l system emr p l o y e d was a s o l u t i o n o f benzophenone ( c o n c e n t r a t i o n such t h a t A355 = 0.5) and n a p h t h a l e n e , N, (0.1M), or b i p h e n y l , B, (0.1M), or f l u o r e n e , F, (0.1M) i n e i t h e r a e r a t e d benzene o r a e r a t e d c y c l o h e x o ane. Under these c o n d i t i o n s the o n l y l i g h t c a b s o r b i n g s p e c i e s i s benzophenone w h i c h , on e x c i t a t i o n , produces i t s t r i p l e t s t a t e w i t h u n i t quantum y i e l d . T h i s then undergoes energy t r a n s f e r t o the n a p h t h a l e n e , e t c . , which i s p r e s e n t a t 0.1M t o ensure 100$ energy

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

RODGERS

Singlet Oxygen Quantum Yields

a

CUVETTE SILICON FILTER

K"///////A

b

i 0.0

40.0

80.0 TIME

120.0 160.0 200.0 (MICROSECONDS)

240.0

F i g u r e 3- ( a ) Schematic o f t i m e - r e s o l v e d i n f r a r e d l u m i n e s c e n c e a p p a r a t u s used f o r o b t a i n i n g i n t e n s i t y - t i m e p r o f i l e s o f s i n g l e t oxygen e m i s s i o n as shown i n ( b ) , which i s such a s i g n a l obt a i n e d from a 355 n m - i r r a d i a t e d , a e r a t e d benzene s o l u t i o n o f 2-acetonaphthane.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

86

LIGHT-ACTIVATED PESTICIDES

t r a n s f e r , whence hydrocarbon t r i p l e t s t a t e s a r e formed w i t h u n i t efficiency. T h i s p r o c e s s i s complete i n l e s s than 1 0 7 s . In aera t e d s o l v e n t , a l l h y d r o c a r b o n t r i p l e t s t a t e s a r e quenched by oxygen and, s i n c e t h e c o n d i t i o n s have been s e l e c t e d such t h a t *T = 1.0, we have r

*A

=

SA'^T

=

S

A

A p l o t o f 1^ V S l a s e r energy has a s l o p e t h a t i s d i r e c t l y p r o p o r t i o n a l t o S and as F i g u r e k shows, i n benzene (upper h a l f ) t h e s l o p e s f o r N, F and B a r e d i s t i n c t l y d i f f e r e n t and hence t h e values d i f f e r . I n c y c l o h e x a n e , however ( l o w e r h a l f ) , i t i s c l e a r t h a t N, F and B have a common s l o p e and t h e r e f o r e a common value. That t h r e e d i s s i m i l a r m o l e c u l e s have a common S strongly i m p l i e s t h a t the common v a l u e i s u n i t y . T h i s was c o n f i r m e d by c a r e f u l l y measuring the c o n c e n t r a t i o n o f N(T<|) produced i m m e d i a t e l y a f t e r a 355 nm l a s e r p u l s i n t solutio containin both benzophenone and n a p h t h a l e n c o n d i t i o n s measuring th b l e a c h e d by t h e 0 ( A ) g e n e r a t e d from the p o p u l a t i o n o f N ( T ^ ) . This confirmed t h a t f o r naphthalene i n cyclohexane, =1.0. A s i m i l a r experiment was used t o show t h a t = 0.55 f o r n a p h t h a l e n e i n benzene. Two o t h e r s e r i e s o f e x p e r i m e n t s showed t h a t i n a c e t a nitrile = 1.0 f o r n a p h t h a l e n e and, i n a medium composed o f sodium d o d e c y l s u l f a t e (SDS) m i c e l l e s i n D 0 c o n t a i n i n g benzophenone and n a p h t h a l e n e , a g a i n S =» 1.0 was o b t a i n e d . I n t h i s way we now have a b s o l u t e s t a n d a r d s f o r S and f u r t h e r , we have a b s o l u t e s t a n dards f o r $ where ketone t r i p l e t s w i t h * j = 1.0 a r e used as p r i mary s e n s i t i z e r s (Gorman, A.A. e t a l , s u b m i t t e d f o r p u b l i c a t i o n ) . As an example suppose we have a m o l e c u l e X o f unknown and $A. I f X absorbs s i g n i f i c a n t l y a t 355 nm i t s *A can be d e t e r m i n e d by measuring I * as a f u n c t i o n o f l a s e r energy f o r a s o l u t i o n o f X i n c y c l o h e x a n e (or any o t h e r known s o l v e n t ) and d o i n g the same f o r a s t a n d a r d benzophenone-naphthalene s o l u t i o n ( i n t h e same s o l v e n t ) . The r a t i o o f the s l o p e s of the two p l o t s i s i d e n t i c a l t o the r a t i o o f the *A v a l u e s f o r X and N and s i n c e t h e l a t t e r i s u n i t y can be c a l c u l a t e d . O t h e r w i s e , where X does not absorb a t 355 nm and E* < E^P, A

A

1

2

G

2

A

A

A

then S* can be measured 1^ from o p t i c a l l y - m a t c h e d s o l u t i o n s cont a i n i n g ( i n the same s o l v e n t ) , (a) benzophenone and n a p h t h a l e n e , and (b) benzophenone and X.

Then we see t h a t

IX A IN A

but

AX

CX

A "

*N A

.

AX

A "

S

N

A

• *N A

= *N = $Bp s i n c e complete energy t r a n s f e r o c c u r s , t h u s :

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

RODGERS

Singlet Oxygen Quantum Yields

l /mV A

0

03

SB

0.9

m

j

F i g u r e 4. I n f r a r e d e m i s s i o n i n t e n s i t y e x t r a p o l a t e d t o t=0 (1^) v e r s u s l a s e r energy f o r (a) a e r a t e d benzene s o l u t i o n s of p-methoxy acetophenone w i t h 0.1M naphthalene ( A ) , b i p h e n y l (o) or f l u o r e n e (o) and (b) c o r r e s p o n d i n g p l o t f o r c y c l o h e x a n e s o l u t i o n s . I n s e r t shows t y p i c a l i n t e n s i t y * - t i m e p r o f i l e i n l a t t e r w i t h T = 23-5 s.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

88

LIGHT-ACTIVATED PESTICIDES

A

A

and s i n c e S^j i s known ( I n benzene, c y c l o h e x a n e , a c e t o n i t r i l e and SDS m i c e l l e s ) then S* i s c a l c u l a t e d . compute *x when *x i

s

Having d e t e r m i n e d S

A

we can

known.

An i m p o r t a n t p o i n t t o remember i n e x p e r i m e n t s such as t h e s e i n which t h e l u m i n e s c e n c e i n t e n s i t y i s used as a measure o f 0 2 ( A ) c o n c e n t r a t i o n i s t h a t t h e i n t e n s i t y o f l u m i n e s c e n c e a t any time i s a product o f the number o f e m i t t i n g s t a t e s and t h e i r r a d i a t i v e probability. T h i s l a s t , u s u a l l y denoted by t h e r a d i a t i v e r a t e c o n s t a n t ( k ) f o r t h e t r a n s i t i o n 1 A ' •••• • > 3z~ p r o b a b l y v a r i e s from s o l v e n t t o s o l v e n t . Thus measurements o f unknown and s t a n d a r d must employ a common s o l v e n t 1

G

R

VALUES OF $

A

G

AND S

A

FOR A VARIETY OF

SENSITIZERS

A summary o f S v a l u e s f o r a number o f p h o t o s e n s i t i z e r s i n a v a r i e t y of s o l v e n t s i s p r e s e n t e d i n T a b l e I . These were o b t a i n e d u s i n g the i n f r a r e d luminescence measurements as d e s c r i b e d i n t h e p r e c e e d i n g s e c t i o n (44_). For many o f t h e s e systems the l i t e r a t u r e con«t a i n s <&T v a l u e s thus e n a b l i n g a c a l c u l a t i o n o f the * t h a t would r e s u l t from d i r e c t e x c i t a t i o n o f t h e p h o t o s e n s i t i z e r . R e c e n t l y , u s i n g t h e r m a l l e n s i n g (37.). R o s s b r o l c h e t a l have c o n f i r m e d t h e anthracene v a l u e s i n T a b l e I and have e x p e r i m e n t a l l y shown t h a t f o r t h i s m o l e c u l e $T = 0.78. They a l s o measured * v a l u e s f o r mesor-tetraphenylporphine and i t s z i n c d e r i v a t i v e s f i n d i n g v a l u e s o f 0.58 and 0.73 r e s p e c t i v e l y . U s i n g l a s e r f l a s h p h o t o l y s i s and t h e DPBF b l e a c h i n g method Chattopadhyay e t a l (19_,20) have determined S v a l u e s f o r a s e r i e s o f a r o m a t i c ketones and some p o l y e n e s . These a r e p r e s e n t e d i n T a b l e I I . The f u r o c o u m a r i n s a r e a c l a s s o f m o l e c u l e s t h a t have s i g n i f i c a n c e i n p h o t o b i o l o g y and $ v a l u e s f o r a s e r i e s of such m o l e c u l e s have been measured (j*2) u s i n g t h e i n f r a r e d l u m i n e s c e n c e method. These d a t a a r e c o l l e c t e d i n T a b l e I I I . Some f u r o c o u m a r i n s show v e r y weak s i n g l e t oxygen p r o d u c t i o n . T h i s appears t o a r i s e m a i n l y from low i n t e r s y s t e m c r o s s i n g y i e l d s ( T a b l e III.) A l l the e v a l u a t i o n s i n T a b l e s I through I I I have been o b t a i n e d u s i n g u l t r a - v i o l e t e x c i t a t i o n , whereas most photodynamic i n t e r e s t c e n t e r s around v i s i b l e - - a b s o r b i n g dyes such as xanthenes, p o r p h y r i n s , p h t h a l o c y a n i n e s , and so f o r t h . Although v i s i b l e - a b s o r b i n g dyes t o some e x t e n t a l s o absorb u.v. r a d i a t i o n and a r e thus measu r a b l e by t h o s e methods used f o r a r o m a t i c ketones, e t c . , u n c e r t a i n t i e s e x i s t as t o whether wavelength e f f e c t s can o c c u r . The t e c h n i q u e s t h a t have been used f o r u v ^ a b s o r b i n g m o l e c u l e s a r e i n p r i n c i p l e t r a n s l o c a t a b l e t o the v i s i b l e . Some r e s t r i c t i o n s occur i n p r a c t i c e , however. F o r example, t h o s e methods t h a t r e l y on t h e t r a n s i e n t b l e a c h i n g o f DPBF1 o r a n t h r a c e n e d i p r o p i o n i c a c i d (21) may n o t be u s e a b l e f o r s e n s i t i z e r s (eg p o r p h y r i n s ) t h a t absorb l i g h t s t r o n g l y near 400 nm where t h e s e probes a r e a l s o a b s o r b i n g . A

A

A

A

A

4

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

5.

RODGERS

89

Singlet Oxygen Quantum Yields

Table I . S

A

Values f o r U-V Absorbing S e n s i t i z e r s i n Several Solvents SOLVENTS Cyclo.hexane

Sensitizer

Benzene

Acetor, nitrile

Aq.SDS Micelles

naphthalene (a)

1.0

0.55

1.0

1.0

biphenyl (a)

1.0

0.42

-rr

1.0

fluorene (a) a c r i d i n e (b)

1.0

benzophenone (b)

rr-r

0.29

rrc

rrr

anthracene (c)

rr-r.

0.8

•

rrr

fluorenone (c)

r.rr

0.8

•-rr

rrr

2-acetonaphthone

0

-rr-

rr-

Γ.-ΓΪ

-rr

.93

cyclopentadiene (d)

(d)

0.7

(b)

0.58

η--

cyclohexadiene (d)

0.66

0.39

cycloheptatriene (d)

0.89

0.69

-rr

rrn

0.45

r~-

r..-r

r.r-r

rrn

tetramethylrbutadiene (d) nea-allociraene (d)

0.56

transnstilbene (e)

Γ"

ergosterol ( f )

rrr

(a) (b) (c) (d) (e) (f)

—

0.43 Γ

0.18 0.78

Gorman, A.A. et a l , to be published reference 13 reference 46 Gorman and Rodgers, to be published reference 47 Gorman, Α.Α.; Hamblett, I ; Rodgers, M.A.J. Photochem. Photobiol. In press

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

rrr

90

LIGHT-ACTIVATED PESTICIDES

Table I I .

Φ Values f o r Aromatic Ketones, Polyene and other S e n s i t i z e r s ^ ) Δ

3

Benzene

Cyclon hexane

Aceton nitrile

Methanol

acetophenone

0.35

n-r

0.52

r%-«r

pr-methoxyacetophenone

0.27

Sensitizer

0.42

pncyanoacetophenone bz*

0.39

npr

0.37

n-rr-

p,p'-bis(N,Nrdimethylamino)bz*

0.41

ΟΡΓ.

0.35

n«n

ρ,ρ'-dimethoxy bz*

0.34

ΠΓΡ

0.40

rv— —.

prfluorobz*

0.43

arr.

0.44

ΠΓ-f-

pntrlfluoromethylbz*

0.42

- - p

0.54

r-n

pyrenerlnaldehyde

0.68

0.87

"PR

nrn

allr:transr retinal

nrr-

0.66

nr.r

0.20

Brapor.l4'-carotenal (b)

nrn

0.48

c^7 aldehyde (°)

r.rr

0.72

r»-.r Γ.ΓΓ

0.42

* bz - benzophenone (a) references 19 and 20, a l l using

DPBJ

bleaching and t r a n s i e n t spectroscopy.

(b) immediate higher homologue of r e t i n a l (C o aldehyde). 2

(c) immediate lower homologue of r e t i n a l .

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987. 0.084 0.30 0.49

0.41 1.0 0.96

4,5,8r-trimethylpsoralen

3-carbethoxypsoralen

3r.carbethoxypseudopsoralen

u s i n g i n f r a r e d luminescence w i t h Φτ measurement by t r a n s i e n t

0.0040

0.10

5,8-dimethoxypsoralen

r e f e r e n c e 42: absorption.

0.0044

0.40

8-methoxypsoralen

(a)

0.021

0.32

5«-methoxypsoralen

0.012

*Δ

0.026

a

V a l u e s f o r some Furocoumarins i n Benzene( )

0.57

and

pseudopsoralen

A

0.3^

S

psoralen

Sensitizer

Table I I I .

92

LIGHT-ACTIVATED PESTICIDES

A l s o the i n f r a r e d luminescence method has not been c a l i b r a t e d f o r v i s i b l e w a v e l e n g t h a b s o r b e r s but t h i s i s c u r r e n t l y underway i n the author's l a b o r a t o r y . Most quantum y i e l d work on the xanthene dyes ( r o s e b e n g a l , e o s i n , e t c . ) has been c a r r i e d out u s i n g t he s t e a d y s t a t e t e c h n i q u e s . Thus Gandin e t a l ( 2 3 ) used both ADPA b l e a c h i n g and t h e RNO method t o e v a l u a t e Φ v a l u e s f o r a s e r i e s of s u b s t i t u t e d f l u o r e s c e i n s i n water (Table I V ) . Lamberts and Neckers (Z4) used the p h o t o s e n s i t i z e d o x i d a t i o n o f 2,3 d i p h e n y l - p - d i o x e n e f o r measuring ΦΔ v a l u e s f o r a s e r i e s o f r o s e bengal d e r i v a t i v e s ( T a b l e V ) . Paczkowski and Neckers (25) used the same method f o r e v a l u a t i n g Φ f o r a s e r i e s o f r o s e bengal-tagged polymers w i t h d i f f e r e n t degrees of l o a d i n g . Because o f the i n t e r e s t i n p o r p h y r i n s i n photodynamic tumor t h e r a p y , many porphine d e r i v a t i v e s have been s u b j e c t e d t o e x p e r i mental e v a l u a t i o n s f o r s i n g l e t oxgyen f o r m a t i o n . Reddi e t a l ( 4 3 ) found Φ v a r i e d betwee v a r y i n g from aqueous m i c e l l e ments i n d i c a t e d t h a t varie Φχ r e c e n t l y (44_) the i n f r a r e d luminescence method has been used t o e v a l u a t e Φ o f s e v e r a l p o r p h y r i n s (355 nm e x c i t a t i o n ) i n benzene s o l u t i o n ( T a b l e V I ) . V e r l h a c e t a l (26) have used t he RNO method t o measure Φ v a l u e s f o r a s e r i e s of w a t e r r - s o l u b l e p o r p h y r i n s and t h e i r m e t a l l o - d e r i v a t i v e s (Table V I I ) . The c o n t e n t s of t h i s s e c t i o n r e p r e s e n t the m a j o r i t y o f the d a t a t h a t i s c u r r e n t l y a v a i l a b l e on s i n g l e t oxygen quantum y i e l d s (Φ ) and on i t s e f f i c i e n c y o f f o r m a t i o n from a s e n s i t i z e r t r i p l e t - s t a t e ( S ) . A s c r u t i n y o f the T a b l e s shows c l e a r l y t h a t photoe x c i t a t i o n o f a s e n s i t i z e r l e a d s t o a quantum e f f i c i e n c y of s i n g l e t oxygen p r o d u c t i o n , the o r d e r o f magnitude o f which depends predomi n a n t l y upon Φ . T h i s i s because S v a l u e s l i e between 0.3 and 1.0 ( t h e o n l y m o l e c u l e o u t s i d e t h i s range i s 5 , 8 dimethoxypsoralen which has S = 0 . 1 .-Table I V ) . Thus the r u l e o f thumb must be: when s e l e c t i n g a s e n s i t i z e r f o r photodynamic work l o o k f o r one w i t h h i g h Φ·ρ. F i n a l l y , r e c a l l i n g t h a t the parameter i n f o r m s on t h e n a t u r e of i n t e r a c t i o n between the m o l e c u l a r t r i p l e t s t a t e and oxygen, we need t o ask what f a c t o r s a r e c a u s i n g v a l u e s t o v a r y i n the broad range 0.3 t o 1 . 0 ? There i s no d e f i n i t i v e answer y e t t o t h i s ques t i o n , but t h e r e a r e i n d i c a t i o n s t h a t f o r some a r o m a t i c ketones (eg benzophenone, acetophenone) the c o l l i s i o n complex i n r e a c t i o n 1 1 ( a - c ) can a l s o f i n d a bond^forming r o u t e t o the ground s t a t e , perhaps i n v o l v i n g a b i r a d i c a l s p e c i e s . More e v i d e n c e s t i l l needs t o be c o l l e c t e d t o s u b s t a n t i a t e t h i s and t o determine whether t h i s p u t a t i v e c h e m i c a l channel e x i s t s f o r o t h e r s e n s i t i z e r s . Δ

Δ

Δ

Δ

Δ

Δ

A

τ

A

A

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

5. RODGERS

93

Singlet Oxygen Quantum Yields

Table IV. Φ

3

Δ

Values from Xanthene Dyes i n Aqueous S o l u t i o n ^ ) Xanthene (b)

Φ

Δ

FlIi|Clj| (°)

0.75

FlBri^Clii

0.65

F1I| FlBri4 (e)

0.57

FlBr (N02) 2

FII

FlBr

3

0.44

FlBr

2

0.42

4',5'F1C1

(b) (c)

0.52 0.48

2

2',7'F1C1

(a)

2

2

0.07

2

0.07

FICljj

0.05

Fl.

0.03

reference 23: by steady-state techniques using ADPA bleaching and RNO method. F l indicates f l u o r e s c e i n skeleton rose bengal (d) erythrosin B (e) eosin Y

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

(b)

(a) 2

r«-r

0.61

0.71

0.67

0.48

0.61

-rn

D ichloromethane

r e f e r e n c e 24: s t e a d y s t a t e i l l u m i n a t i o n , d i p h e n y l n p - d i o x e n e photocon version. Rj i s a t c a r b o x y l a t e ; R i s a t phenoxide. See o r i g i n a l paper (24) f o r structural features.

Acetyl

Et

3

Et NH

Et

0.74

0.74

Et NH

PhCh 3

3

2

0.72

Et NH

Et NH

3

0.73

H

Ethyl(Et)

Methanol

0.76

residue^)

Values Obtained f o r a S e r i e s of D e r i v a t i v e s of Rose B e n g a l ^ )

Na

2

Δ

R

Φ

Na

Rl r e s i d u e ^ )

T a b l e V.

5.

95

Singlet Oxygen Quantum Yields

RODGERS

Table V I .

S

A

and Φ Values f o r some P o r p h y r i n s i n Benzene S o l u t i o n ^ ) Δ

3

Porphyrin

protoporphyrin dimethylester

0.71

0.57

hemat©porphyrin d i m e t h y l e s t e r

0.70

0.50

mesoporphyrin d i m e t h y l e s t e r

0.70

0.57

deuteroporphyrin

0.78

0.56

0.74

0.49

dimethylester

photoprotoporphyrin

dimethylester

(a)

by i n f r a r e d l u m i n e s c e n c e measurements.

r e f e r e n c e 44:

Table V I I . Φ Values f o r W a t e r - S o l u b l e P o r p h y r i n s and Métallo D e r i v a t i v e s i n Water a t ρ Η = 7 ^ Δ

Porphyrin

mesa-tetra

Φ

( 4 r N o m e t h y l p y r i d y l ) porphine

m e s o n t e t r a (4r-carboxyphenyl) mesor-.tetra ( 4 - s u l f o n a t o p h e n y l ) meso-tetra

(TMPy)

0.74 0.58

porphine

0.62

porphine

(4<-N,Ν',Ν " - t r i m e t h y l a m i n o p h e n y l )

Δ

porphine

0.77

hematoporphyrin

0.22

zincrtTMPyP

0.88

magnesium - TMPyP

0.69

cadmium π TMPyΡ

0.75

p a l l a d i u m « TMPyP

0.12

copper ( I I ) «- TMPyP

<10"3

c o b a l t - TMPyP

<10<-3

manganese π TMPyP

<10^3

(a)

r e f e r e n c e 26:

u s i n g RNO method.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

LIGHT-ACTIVATED PESTICIDES

96

Acknowledgments The e n t h u s i a s t i c c o l l a b o r a t i o n and s u p p o r t o f Dr. A. A. Gorman i n a l l a s p e c t s o f t h e a u t h o r ' s s i n g l e t oxygen a c t i v i t i e s a r e most g r a t e f u l l y acknowledged. The Center f o r F a s t K i n e t i c s Research i s s u p p o r t e d j o i n t l y by NIH Grant RR 00886 from the B i o m e d i c a l Re s e a r c h Technology Branch o f t h e D i v i s i o n o f Research Resources and by t h e U n i v e r s i t y o f Texas a t A u s t i n . Research e f f o r t s on s i n g l e t oxygen a r e s u p p o r t e d i n p a r t by NIH Grant GM 24235.

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

Arnold, S . J . ; Kubo, J.; Ogryzlo, E.A. In Advan. Chem. Ser., 77, 133, 1968. Krasnovsky, A.A. Chem. Phys. Lett., 1981, 81, 443. Matheson, I.B.C.; Lee, J . Chem. Phys. Lett. 1972, 14, 350. Birks, J.B. Photophysic f Aromati Molecules Wiley Ne York, 1970. Spikes, J.B. In Th i Plenum: New York, 1977. The Science of Photomedicine; Regan, J . D . ; Parrish, J . Α . , Eds.; Plenum: New York. Brauer, H.D.; Acs. A.; Drew, W.; Gabriel, R.; Ghaeni, S.; Schmidt, R. J . Photochem. 1984, 25, 475. Stevens, B.; Ors, J.A. J . Phys. Chem. 1986, 80, 2164. Wu, K.C.; Trozzolo, A.M. J . Phys. Chem., 1979, 83, 3180. Gijzeman, O . L . J . ; Kaufman, F . ; Porter, G. J . Chem. Soc. Faraday Trans. II, 1973, 69, 708. Gorman, Α.Α.; Lovering, G.; Rodgers, M.A.J. J . Amer. Chem. Soc. 1978, 100, 4527. Garner, Α.; Wilkinson, F. In Singlet Oxygen; Ranby, B.; Rabek, J . F . , Eds.; Wiley: New York, 1978; p. 48. Gorman, Α.Α.; Hamblett; I . ; Rodgers, M.A.J. J . Amer. Chem. Soc., 1984, 106, 4679. Adams, D.R.; Wilkinson, F. J . Chem. Soc., Faraday Trans. II, 1972, 68, 586. Merkel, P.B.; Kearns, D.R. Chem. Phys. Lett. 1971, 12, 120. Young, R.H.; Brewer, D.; Keller, R.A. J . Amer. Chem. Soc. 1973, 905, 375. Gorman, Α.Α.; Lovering, G.; Rodgers, M.A.J. Photochem. Photo biol. 1976, 23, 299. Ramesh, V.; Ramnath, N.; Ramamurthya, V. J . Photochem. 1982, 18, 293. Chattopadhyay, S.K.; Kumar, C.V.; Das, P.K. J . Photochem. 1984, 18, 293. Chattopadhyay, S.K.; Kumar, C.V.; Das, P.K. J . Photochem. 1985, 30, 81. Singh, Α.; McIntyre, N.R.; Koroll, G.W. Photochem. Photobiol. 1978, 28, 595. Lindig, B.A.; Rodgers, M.A.J.; Schaap, A.P. J . Am. Chem. Soc. 1980, 102, 5590. Gandin, E . : Lion, Y.; Van de vorst, A. Photochem. Photobiol. 1983, 37, 271. Lamberts, J . J . M . ; Neckers, D.C.; Tetrahedron, 1985, 41, 2183.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

5. RODGERS 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47.

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97

Paczkowski, J.; Neckers, D.C.; Macromolecules, 1985, 18, 1245. Verlhac, J . B . ; Gandemer, Α.; Kraljic, I. Nouv. J . Chim. 1984, 8, 401. Young, R.H.; Brewer, D.R. In Singlet Oxygen; Ranby, B.; Rabek, J . F . , Eds.; Wiley: New York, 1978, p. 36. Krasnovski, A.A. Photochem. Photobiol. 1979, 29, 29. Byteva, I.M.; Gurinovitch, G.P. J . Lumin. 1979, 21, 17. Salakhiddinov, K . I . ; Byteva, I.M.; Dzhagarev, B.M. Opt. Spectrosk. 1979, 47, 881. Khan, A.U.; Kasha, M. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 6047. Hurst, J . R . ; McDonald, J . D . ; Schuster, G.B. J . Am. Chem. Soc. 1982, 104, 2065. Parker, J . G . ; Stanbro, W.D. J . Am. Chem. Soc. 1982,104 Ogilby, P.R.; Foote, C.S. J . Am. Chem. Soc. 1982, 104, 2069. Rodgers, M.A.J.; Snowden Fuke, K.; Ueda, M. 372. Rossbroich, G.; Garcia, N.A.; Braslavsky, S.E. J . Photochem. 1985, 31, Khan, A.U. This Volume; Chapter XX. Khan, A.U. Chem. Phys. Lett. 1980, 72, 112. Kanofsky, J.R. J . Biol. Chem. 1983, 258, 5991. Rodgers, M.A.J. J . Am. Chem. Soc. 1983, 105, 6201. Knox, C.N.; Land, E.J.; Truscott, T.G. Photochem. Photobiol. 1986, 43, 359. Reddi, E . ; Jori, G; Rodgers, M.A.J.; Spikes, J.D. Photochem. Photobiol. 1983, 38, 639. Keene, J . P . ; Kessel, D.; Land, E . J . ; Redmond, R.W.; Truscott, T.G. Photochem. Photobiol. 1986, 43, 117. Gorman, Α.Α.; Rodgers, M.A.J. J . Am. Chem. Soc. 1986, 108, 5074. Gorman, Α.Α.; Hamblett, I.; Rodgers, M.A.J. J . Photochem. 1984, 25, 115. Gorman, Α.Α.; Rodgers, M.A.J. Chem. Phys. Lett. 1984, 120, 58.

RECEIVED November 20, 1986

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 6

Biochemistry of Photodynamic Action 1

2

John D. Spikes and Richard C. Straight 1

Department of Biology, University of Utah, Salt Lake City, UT 84112 Veterans Administration Medical Center and Departments of Medicine and Surgery (Laser Institute), University of Utah School of Medicine, Salt Lake City, UT 84148

2

Cells and organism exposure to ligh sensitizers. This phenomenon, which typically requires molecular oxygen, is termed photodynamic action and results from the sensitized photodegradation of essential biomolecules in cells. The major cellular targets are unsaturated lipids, proteins and nucleic acids. Photodynamic effects are mediated by photochemically generated singlet oxygen, free radicals, hydrogen peroxide and superoxide, depending on the sensitizer, the chemical nature of the molecule being photodegraded, and the reaction conditions. This paper reviews the photochemical and biochemical pathways involved in the photodynamic degradation of the major classes of susceptible biomolecules.

Organisms are v e r y s e n s i t i v e t o s h o r t e r wavelength u l t r a v i o l e t r a d i a t i o n t h a t i s absorbed e f f i c i e n t l y by e s s e n t i a l b i o m o l e c u l e s such as n u c l e i c a c i d s and p r o t e i n s . R a d i a t i o n i n the l o n g wavelength u l t r a v i o l e t - v i s i b l e range i s much l e s s h a r m f u l , i n p a r t because r e l a t i v e l y few types o f b i o m o l e c u l e s absorb a p p r e c i a b l y i n t h i s r e g i o n of the spectrum. However, i n the presence o f a p p r o p r i a t e p h o t o s e n s i t i z e r s , a l l k i n d s o f organisms, p l a n t and a n i m a l , s i n g l e c e l l e d and m u l t i c e l l u l a r , are i n j u r e d and k i l l e d by l i g h t i n t h i s range (1,2) · The h a r m f u l e f f e c t s r e s u l t from the s e n s i t i z e d p h o t o a l t e r a t i o n o f c r i t i c a l types of b i o m o l e c u l e s ; t h i s i n t u r n i n t e r f e r e s w i t h m e t a b o l i c processes and the proper f u n c t i o n of c e l l u l a r s t r u c t u r e s and o r g a n e l l e s ( c e l l membranes, m i t o c h o n d r i a , nuclei, etc.). T h i s chapter reviews the b i o c h e m i c a l changes t h a t occur i n d i f f e r e n t k i n d s o f molecules o f b i o l o g i c a l importance as a r e s u l t o f i l l u m i n a t i o n i n the presence o f p h o t o s e n s i t i z e r s . P r o b a b l y the f i r s t b i o c h e m i c a l study o f a photodynamic r e a c t i o n was t h a t o f P r o f e s s o r Hermann von Tappeiner and h i s s t u d e n t s who showed i n 1903

0097-6156/87/0339-0098$06.00/0 © 1987 American Chemical Society

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t h a t e o s i n s e n s i t i z e s the p h o t o i n a c t i v a t i o n of the enzymes d i a s t a s e , i n v e r t a s e and p a p a i n under a e r o b i c c o n d i t i o n s (3)· Von T a p p e i n e r c o i n e d the term "photodynamic" i n a c t i v a t i o n f o r t h i s phenomenon (^_) · Much l a t e r the i n a c t i v a t i o n was shown to r e s u l t from the s e n s i t i z e d p h o t o o x i d a t i o n of c e r t a i n amino a c i d r e s i d u e s i n the p r o t e i n s (_5) · Blum ( 4 ) , i n h i s s e m i n a l monograph "Photodynamic A c t i o n and D i s e a s e s Caused by L i g h t " , suggested t h a t the term "photodynamic a c t i o n " be c o n f i n e d to those p h o t o s e n s i t i z e d r e a c t i o n s t h a t r e q u i r e m o l e c u l a r oxygen and i n which oxygen i s consumed. Not a l l i n v e s t i g a t o r s accept t h i s d e f i n i t i o n , but f o r c o n v e n i e n c e , we w i l l use i t i n t h i s r e v i e w . A c t u a l l y , most p h o t o s e n s i t i z e d r e a c t i o n s i n v o l v i n g b i o m o l e c u l e s do r e q u i r e m o l e c u l a r oxygen. There are n o t a b l e e x c e p t i o n s , however, which cannot be covered i n t h i s r e v i e w (_1) · Comments on Photodynamic Mechanisms and

Photosensitizers

Two p r i n c i p l e mechanism termed Type I and Type I p r o c e s s e s , r e s p e c t i v e l y ( 6 - 9 ) Typ r e a c t i o n s the l i g h t - e x c i t e d s e n s i t i z e r ( t y p i c a l l y i n i t s t r i p l e t s t a t e ) r e a c t s d i r e c t l y w i t h the s u b s t r a t e v i a an e l e c t r o n or hydrogen atom t r a n s f e r w i t h the p r o d u c t i o n of f r e e r a d i c a l forms of the two r e a c t a n t s . These s p e c i e s can r e a c t f u r t h e r i n a number of ways; i n the presence of oxygen the p r o d u c t s are o f t e n an o x i d i z e d form of the s u b s t r a t e and the r e g e n e r a t e d ground s t a t e of the s e n s i t i z e r . I n some r e a c t i o n s of t h i s type, s u p e r o x i d e or hydrogen p e r o x i d e i s produced which can o x i d i z e some b i o m o l e c u l e s . S e n s i t i z e r t r i p l e t i n Type I I r e a c t i o n s i n t e r a c t s by energy t r a n s f e r w i t h ground s t a t e oxygen ( w h i c h i s a t r i p l e t ) y i e l d i n g ground s t a t e s e n s i t i z e r and s i n g l e t oxygen; t h i s l a t t e r s p e c i e s i s h i g h l y e l e c t r o p h i l i c and can r e a c t w i t h many types of b i o m o l e c u l e s much more r a p i d l y than does ground s t a t e oxygen ( 6 - 9 ) . A l t h o u g h photodynamic r e a c t i o n s i n v o l v i n g b i o m o l e c u l e s sometimes appear to be s i m p l e , they more o f t e n t u r n out to be r a t h e r complex. S e v e r a l , o f t e n competing, r e a c t i o n pathways can be i n v o l v e d , depending on the s u b s t r a t e (compound being p h o t o o x i d i z e d ) , the s e n s i t i z e r , the s o l v e n t , and the r e a c t i o n c o n d i t i o n s ( r e a c t a n t c o n c e n t r a t i o n s , pH, s o l v e n t , e t c . ) . I n many cases the p r i m a r y product i s u n s t a b l e and decays v e r y r a p i d l y to secondary p r o d u c t s . A l s o , the p r i m a r y or subsequent p r o d u c t s may themselves be s u s c e p t i b l e to f u r t h e r photodynamic d e g r a d a t i o n ( 6 ) . S e v e r a l hundred compounds have been examined f o r t h e i r a b i l i t y t o s e n s i t i z e p h o t o r e a c t i o n s of b i o m o l e c u l e s . Effective p h o t o s e n s i t i z e r s f o r b i o l o g i c a l systems i n c l u d e n a t u r a l p r o d u c t s such as i r o n - f r e e p o r p h y r i n s ( c o p r o p o r p h y r i a protoporphyrin, u r o p o r p h y r i n ) , f l a v i n s such as r i b o f l a v i n and FMN, c h l o r o p h y l l and a number of o t h e r compounds found i n p l a n t s i n c l u d i n g a l k a l o i d s , extended q u i n o n e s , f u r a n o c o u m a r i n s , p o l y a c e t y l e n e s , t h l o p h e n e s , e t c . A l a r g e number of s y n t h e t i c compounds i n c l u d i n g a c r i d i n e s such as a c r i d i n e orange, a n t h r a q u i n o n e s , a z i n e dyes, many k e t o n e s , a l a r g e number of s y n t h e t i c p o r p h y r i n s , p h t h a l o c y a n i n e s , t h i a z i n e dyes such as methylene b l u e , xanthene dyes such as e o s i n and rose b e n g a l , etc. (2,10).

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100 B i o c h e m i s t r y of Photodynamic R e a c t i o n s

In S o l u t i o n

B i o m o l e c u l e s s u s c e p t i b l e to photodynamic a c t i o n i n c l u d e c e r t a i n o r g a n i c a c i d s , a l c o h o l s , amines, c a r b o h y d r a t e s , n i t r o g e n h e t e r o c y c l i c s , n u c l e i c a c i d s and c e r t a i n n u c l e i c a c i d bases, some p l a n t hormones, p r o t e i n s and c e r t a i n amino a c i d s , p y r r o l e s , s t e r o i d s , u n s a t u r a t e d l i p i d s , some v i t a m i n s , e t c . A v e r y l a r g e amount of r e s e a r c h has been done on the e f f e c t s of photodynamic treatment i n v i t r o ( i n s o l u t i o n ) on these types of b i o m o l e c u l e s , as reviewed b r i e f l y i n the f o l l o w i n g s e c t i o n s . There are s e v e r a l r e c e n t s h o r t reviews of photodynamic e f f e c t s on b i o m o l e c u l e s (1,2,7,8) and one d e t a i l e d r e v i e w (5); these may be c o n s u l t e d f o r a more i n - d e p t h i n t r o d u c t i o n t o the l i t e r a t u r e of t h i s a r e a . Photodynamic E f f e c t s on A l c o h o l s and C a r b o h y d r a t e s . A l c o h o l s and c a r b o h y d r a t e s , i n c l u d i n g s i m p l e s u g a r s , p o l y s a c c h a r i d e s such as c e l l u l o s e , and complex c a r b o h y d r a t e s such as a l g i n a t e s h e p a r i n and h y a l u r o n i c a c i d are p h o t o o x i d i z e s e n s i t i z e r s are the mos t y p i c a l l y proceed by a Type I p r o c e s s . For example, a hydrogen atom i s a b s t r a c t e d from the a l p h a - c a r b o n of a l c o h o l s ; the r e s u l t i n g a l c o h o l r a d i c a l r e a c t s w i t h oxygen, g i v i n g an aldehyde or c a r b o x y l i c a c i d f o r primary a l c o h o l s and a ketone f o r secondary a l c o h o l s (6)· H e x i t o l s are p h o t o o x i d i z e d f i r s t to the hexose and then to the c o r r e s p o n d i n g h e x o n i c a c i d . C e l l u l o s e i s p h o t o o x i d i z e d by s i m i l a r p r o c e s s e s g i v i n g damaged and weakened f i b e r s ( " p h o t o t e n d e r i n g " ) (6)· H y a l u r o n i c a c i d , a h i g h m o l e c u l a r weight g l y c o s a m i n o g l y c a n , i s a major component of the j e l l y - l i k e ground substance of animal t i s s u e s and of the v i t r e o u s humor of the eye. The v i s c o s i t y of h y a l u r o n i c a c i d s o l u t i o n s p r o g r e s s i v e l y decreases on i l l u m i n a t i o n i n the presence of photodynamic s e n s i t i z e r s ; t h i s has g e n e r a l l y been regarded as r e s u l t i n g from a f r e e r a d i c a l i n i t i a t e d s c i s s i o n of the h y a l u r o n i c a c i d c h a i n . Methylene b l u e s e n s i t i z e s a v i s c o s i t y decrease i n a r e a c t i o n a p p a r e n t l y mediated by s i n g l e t oxygen; the change i n v i s c o s i t y appears t o r e s u l t from a l t e r a t i o n s i n the t e r t i a r y s t r u c t u r e of the h y a l u r o n i c a c i d f o l l o w e d by o n l y minor d e p o l y m e r i z a t i o n ( 1 1 ) . C l e a r l y much remains t o be l e a r n e d about the mechanisms of photodynamic e f f e c t s on complex c a r b o h y d r a t e s . Photodynamic E f f e c t s on L i p i d s . The l i p i d s are a d i v e r s e group of compounds i n c l u d i n g f a t t y a c i d s , f a t s ( t r i g l y c e r i d e s of f a t t y a c i d s ) , p h o s p h o l i p i d s , s t e r o i d s and t h e i r d e r i v a t i v e s , e t c . U n s a t u r a t e d f a t t y a c i d s , and f a t s and p h o s p h o l i p i d s c o n t a i n i n g u n s a t u r a t e d f a t t y a c i d s , are s u s c e p t i b l e to photodynamic a c t i o n by both Type I and Type I I p r o c e s s e s (_5) · A l l y l i c h y d r o p e r o x i d e s are formed i n i t i a l l y i n both mechanisms; however, the k i n e t i c s of p h o t o o x i d a t i o n and the d i s t r i b u t i o n of product isomers are d i f f e r e n t i n the two c a s e s . The Type I process i s more complex, g i v i n g r i s e to a l l y l i c f r e e r a d i c a l s t h a t can r e a c t to g i v e d i f f e r e n t h y d r o p e r o x i d e s as w e l l as a l c o h o l s , epoxides and k e t o n e s . The r e a c t i o n w i t h s i n g l e t oxygen i s o f t e n much s i m p l e r , w i t h fewer p r o d u c t s being formed. P o l y u n s a t u r a t e d f a t s and p h o s p h o l i p i d s appear to be p h o t o o x i d i z e d l a r g e l y by a Type I I r e a c t i o n g i v i n g mono- and d i h y d r o p e r o x i d e s ; these i n i t i a l products can undergo dark

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a u t o x i d a t i o n w i t h f u r t h e r d e g r a d a t i o n and the f o r m a t i o n of more complex m i x t u r e s of r e a c t i o n p r o d u c t s (5-7,12)* The i n i t i a l step i n the Type I I process i s an "ene" r e a c t i o n ( 1 3 ) . C h o l e s t e r o l g i v e s a c h a r a c t e r i s t i c a l l y d i f f e r e n t p a t t e r n of p r o d u c t s depending on whether i t i s p h o t o o x i d i z e d by a Type I or a Type I I p r o c e s s . A g a i n , the s i n g l e t oxygen pathway g i v e s a s i m p l e r product p a t t e r n , i . e . , almost e n t i r e l y the 5-alpha-hydroperoxide w i t h o n l y s m a l l amounts of o t h e r h y d r o p e r o x i d e s . In c o n t r a s t , i n the f r e e r a d i c a l p r o c e s s , the 7-alpha- and 7-beta-hydroperoxides are formed along w i t h a number of o t h e r p r o d u c t s ( 5 - 7 , 1 4 ) . Other s t e r o i d s can a l s o be p h o t o o x i d i z e d , i n c l u d i n g p r e d n i s o l o n e , d e o x y c o r t i c o s t e r o n e and s u b s t i t u t e d h y d r o c o r t i s o n e s . For example, they are p h o t o o x i d i z e d t o c a r b o x y l i c a c i d d e r i v a t i v e s on i l l u m i n a t i o n i n the presence of f l a v i n s ( 1 5 ) . E s t r o n e i s i r r e v e r s i b l y photobound t o p r o t e i n i n a s e n s i t i z e d r e a c t i o n ( 1 6 ) , w h i l e some c o n t r a c e p t i v e s t e r o i d s are decomposed by photodynamic treatment ( 1 7 ) . These r e a c t i o n shown by some i n d i v i d u a l Photodynamic E f f e c t s on Amino A c i d s . Of the a p p r o x i m a t e l y 20 amino a c i d s t h a t occur i n p r o t e i n s , o n l y f i v e ( c y s t e i n e , a t h i o l ; h i s t i d i n e , an i m i d a z o l e ; m e t h i o n i n e , an o r g a n i c s u l f i d e ; t r y p t o p h a n , an i n d o l e ; and t y r o s i n e , a phenol) are p h o t o o x i d i z e d r a p i d l y w i t h photodynamic s e n s i t i z e r s ; these amino a c i d s a l l have e l e c t r o n - r i c h s i d e c h a i n s ( 5 , 6 - 8 ) . The mechanisms and k i n e t i c s of amino a c i d p h o t o o x i d a t i o n depend on the amino a c i d , the s e n s i t i z e r , the s o l v e n t , the pH, e t c . (5)· W i t h e o s i n , rose b e n g a l , p r o f l a v i n and p o r p h y r i n s , c y s t e i n e i s p h o t o o x i d i z e d l a r g e l y to c y s t i n e i n a Type I I p r o c e s s ; the pH dependence of the r a t e i n d i c a t e s t h a t the unprotonated t h i o l group i s most r e a c t i v e ( 5 , 1 8 ) . The t h i o l f r e e r a d i c a l i s produced d u r i n g the h e m a t o p o r p h y r i n - s e n s i t i z e d p h o t o o x i d a t i o n of c y s t e i n e ( 1 9 ) . I n c o n t r a s t , c y s t e i n e i s p h o t o o x i d i z e d to c y s t e i c a c i d w i t h c r y s t a l v i o l e t i n what appears t o be a Type I r e a c t i o n ( 2 0 ) . H i s t i d i n e i s r a p i d l y p h o t o o x i d i z e d w i t h many s e n s i t i z e r s . The r e a c t i o n r a t e i n c r e a s e s w i t h pH i n a f a s h i o n demonstrating t h a t the unprotonated i m i d a z o l e r i n g of h i s t i d i n e i s the r e a c t i v e s i t e (5_) · H i s t i d i n e and r e l a t e d i m i d a z o l e s appear to be p h o t o o x i d i z e d by a Type I I process w i t h the f o r m a t i o n of endoperoxides; these are v e r y u n s t a b l e , and break down r a p i d l y w i t h d e s t r u c t i o n of the i m i d a z o l e r i n g and the f o r m a t i o n of a number of secondary products ( 7 , 2 1 ) . W i t h some s e n s i t i z e r s , such as rose b e n g a l , methionine i s p h o t o o x i d i z e d d i r e c t l y to methionine s u l f o x i d e by a Type I I process at low pH, or i f the amino group i s b l o c k e d . At h i g h pH w i t h the amino group f r e e , the p r i m a r y product i s dehydromethionine, which s l o w l y h y d r o l y z e s w i t h the f o r m a t i o n of methionine s u l f o x i d e i n a r a t h e r complex r e a c t i o n ( 1 8 , 2 2 ) . I n c o n t r a s t , w i t h f l a v i n and benzophenone type s e n s i t i z e r s , methionine i s deaminated and d e c a r b o x y l a t e d by a Type I r e a c t i o n t o g i v e the aldehyde, m e t h i o n a l ( 2 3 ) . T h i s can be f u r t h e r photodegraded t o y i e l d a v a r i e t y of products. Tryptophan g i v e s complex m i x t u r e s of p r o d u c t s on p h o t o o x i d a t i o n i n r e a c t i o n s t h a t depend on the s e n s i t i z e r and the r e a c t i o n c o n d i t i o n s ; some of the p r o d u c t s can be f u r t h e r p h o t o o x i d i z e d . Both

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Type I and Type I I p r o c e s s e s can a p p a r e n t l y be i n v o l v e d . One product produced by r e a c t i o n w i t h s i n g l e t oxygen i s Nf o r m y l k y n u r e n i n e , which i t s e l f i s a good photodynamic s e n s i t i z e r (5,24,25). T y r o s i n e appears to be p h o t o o x i d i z e d by both Type I and Type I I r e a c t i o n s ; the r e a c t i o n r a t e i n c r e a s e s w i t h pH i n a manner showing t h a t the phenolate a n i o n i s the most e a s i l y p h o t o o x i d i z e d form of the m o l e c u l e . R e l a t i v e l y l i t t l e i s known of the p h o t o o x i d a t i o n p r o d u c t s , a l t h o u g h r u p t u r e of the t y r o s i n e r i n g does occur (5,6) · A r g i n i n e and l y s i n e have been r e p o r t e d to be p h o t o o x i d i z e d under some c o n d i t i o n s ( 5 ) , and the p h o t o o x i d a t i o n of p h e n y l a l a n i n e i s s e n s i t i z e d by f l a v i n e s (26,27). A l t h o u g h p r i m a r y amine groups are a p p a r e n t l y not p h o t o o x i d i z e d , the c o n c e n t r a t i o n of f r e e amino groups i n a s o l u t i o n of some amino a c i d s decreases on p h o t o o x i d a t i o n ; t h i s may r e s u l t from i n t r a - or i n t e r - m o l e c u l a r dark r e a c t i o n s between i n i t i a l o x y g e n a t i o n products and primary amino groups ( 2 8 ) . Photodynamic E f f e c t s on P r o t e i n s p e r o x i d a s e and s u p e r o x i d , protein t h a t have been examined are s u s c e p t i b l e to photodynamic treatment ( 5^. P r o t e i n s examined i n c l u d e enzymes of a l l c a t e g o r i e s , b l o o d plasma p r o t e i n s ( a l b u m i n s , c e r u l o p l a s m l n , complement, f i b r i n o g e n , hemopexin, heraocyanin, i m m u n o g l o b u l i n s , e t c . ) , hormones ( i n s u l i n , g l u c a g o n , e t c . ) , and m i s c e l l a n e o u s p r o t e i n s such as b a c t e r i a l t o x i n s , c o l l a g e n , cytochromes, eye l e n s c r y s t a l l i n e , g l o b i n s , ovalbumin, o v o t r a n s f e r r i n , snake venom p r o t e i n s , s p e c t r i n , t u b u l i n , e t c . ( a d e t a i l e d l i s t i n g i s g i v e n i n r e f . 5) · Oxygen i s consumed i n these r e a c t i o n s ; however, l i t t l e i s known of i t s u l t i m a t e f a t e i n most c a s e s . The s i t e ( s ) of damage on the p r o t e i n molecule i s t y p i c a l l y one or more of the c y s t e i n y l , h i s t i d y l , m e t h i o n y l , t r y p t o p h y l and t y r o s y l r e s i d u e s (5)· Susceptible residues located at the s u r f a c e of p r o t e i n m o l e c u l e s w i l l tend t o be p h o t o o x i d i z e d f a s t e r than r e s i d u e s b u r i e d i n the i n t e r i o r of the p r o t e i n . I f the p r o t e i n i s c o m p l e t e l y u n f o l d e d , a l l of the s u s c e p t i b l e r e s i d u e s can be p h o t o o x i d i z e d ( 2 9 ) . In most cases p e p t i d e bonds are not broken as a r e s u l t of photodynamic treatment (5_) · Some s e l e c t i v i t y of r e s i d u e p h o t o o x i d a t i o n can be o b t a i n e d u s i n g s e n s i t i z e r s t h a t b i n d t o s p e c i f i c s i t e s on p r o t e i n molecules ( 5 , 3 0 ) . A few p r o t e i n s c o n t a i n n a t u r a l p h o t o s e n s i t i z i n g chromophores bound i n p a r t i c u l a r r e g i o n s ( 5 ) . For example, i n the case of the enzyme 6-phosphogluconate dehydrogenase, the enzyme c o f a c t o r , p y r i d o x a l , s e n s i t i z e s the photodynamic m o d i f i c a t i o n of a h i s t i d i n e r e s i d u e l o c a t e d near the p y r i d o x a l b i n d i n g s i t e of the enzyme ( 3 1 ) . A number of types of p h y s i c o c h e m i c a l changes are observed i n p h o t o d y n a m i c a l l y - t r e a t e d p r o t e i n s i n c l u d i n g a l t e r a t i o n s of a b s o r p t i o n spectrum, a g g r e g a t i o n p r o p e r t i e s , c o f a c t o r - and m e t a l binding p r o p e r t i e s , conformation, mechanical p r o p e r t i e s , o p t i c a l r o t a t i o n , s o l u b i l i t y , v i s c o s i t y , e t c . ( 5 ) . I n c r e a s e d s e n s i t i v i t y to d e n a t u r a t i o n by heat or u r e a , and i n c r e a s e d s u s c e p t i b i l i t y to d i g e s t i o n by p r o t e a s e s are o f t e n observed as a r e s u l t of the photodynamic treatment of p r o t e i n s (32) · The n a t u r e and e x t e n t of a l l of these p h y s i c o c h e m i c a l changes depends on the p r o t e i n , the s e n s i t i z e r , the r e a c t i o n c o n d i t i o n s , the degree of p h o t o o x i d a t i o n of

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the p r o t e i n , e t c . (5)· I l l u m i n a t i o n of some s e n s i t i z e r - p r o t e i n c o m b i n a t i o n s r e s u l t s i n the f o r m a t i o n of c o v a l e n t s e n s i t i z e r - p r o t e i n photoadducts, presumably by Type I p r o c e s s e s ( 5 , 3 3 ) . I n some c a s e s , p r o t e i n s are c r o s s - l i n k e d by photodynamic treatment g i v i n g p r o t e i n d i m e r s , t r i m e r s , e t c . The mechanisms of c r o s s - l i n k i n g are not f u l l y u n d e r s t o o d . I n the case of s p e c t r i n , a membrane p r o t e i n from e r y t h r o c y t e s , i t has been suggested t h a t c r o s s - l i n k i n g r e s u l t s from the i n t e r a c t i o n of a p h o t o o x i d i z e d h i s t i d y l r e s i d u e on one s p e c t r i n m o l e c u l e w i t h a f r e e amino group on another s p e c t r i n m o l e c u l e (34) . The p h o t o s e n s i t i z e d c o v a l e n t c r o s s - l i n k i n g of p r o t e i n s t o DNA (35) , and the p h o t o s e n s i t i z e d c o v a l e n t c o u p l i n g of p r o t e i n s t o s m a l l m o l e c u l e s such as t r y p t o p h a n (36) a l s o o c c u r . Photodynamic treatment u s u a l l y a l t e r s or d e s t r o y s the normal b i o l o g i c a l f u n c t i o n of p r o t e i n s . For example, almost a l l enzymes l o s e t h e i r b i o c a t a l y t i c a c t i v i t y as a r e s u l t of the d e s t r u c t i o n of e s s e n t i a l amino a c i d r e s i d u e s i n the a c t i v e s i t e or b i n d i n g s i t e of the enzyme or by the a l t e r a t i o are n e c e s s a r y f o r the norma (_5). The a n t i g e n i c i t y of some p r o t e i n s as w e l l as t h e i r a b i l i t y t o r e a c t w i t h a n t i b o d i e s d i r e c t e d toward them i s d e c r e a s e d . The b i o l o g i c a l f u n c t i o n s of p e p t i d e hormones such as a n g i o t e n s i n , g l u c a g o n , i n s u l i n , e t c . are d e s t r o y e d by photodynamic t r e a t m e n t , and p r o t e i n t o x i n s from b a c t e r i a , p l a n t s and snakes are i n a c t i v a t e d (5_). Photodynamic E f f e c t s on P u r i n e s and P y r i m i d l n e s . The s e n s i t i z e d p h o t o o x i d a t i o n of b i o l o g i c a l l y important p u r i n e s and p y r i m i d l n e s , t h e i r n u c l e o s i d e s and n u c l e o t i d e s , and some r e l a t e d compounds has been examined u s i n g a number of d i f f e r e n t s e n s i t i z e r s ( 5 , 3 7 ) . In g e n e r a l , under p h y s i o l o g i c a l c o n d i t i o n s , p u r i n e s are p h o t o o x i d i z e d more r e a d i l y than p y r i m i d l n e s , and guanine and i t s d e r i v a t i v e s are more s u s c e p t i b l e than o t h e r p u r i n e s (5_). Ring s u b s t i t u e n t s on n u c l e i c a c i d bases can have a major e f f e c t on the s e n s i t i v i t y t o photooxidation. For example, w i t h hematoporphyrin, 5 - a m i n o u r a c i l and 5 - h y d r o x y u r a c i l are p h o t o o x i d i z e d v e r y much f a s t e r than 5raethyluracil ( 3 8 ) . The p h o t o o x i d a t i o n of guanine and some of the o t h e r n u c l e i c a c i d bases can o c c u r by both Type I and Type I I p r o c e s s e s , w i t h the r e l a t i v e involvement of each pathway depending on the p h o t o s e n s i t i z e r used. Rose bengal s e n s i t i z e s the p h o t o o x i d a t i o n of 3 * , 5 - d i - 0 - a c e t y l - 2 - d e o x y g u a n o s i n e l a r g e l y by a Type I I mechanism; i n c o n t r a s t , r i b o f l a v i n and benzophenone s e n s i t i z e the r e a c t i o n p r i m a r i l y by a Type I process ( 3 9 ) . R i b o f l a v i n a l s o s e n s i t i z e s the p h o t o o x i d a t i o n of 2'-deoxyguanosine by a Type I p r o c e s s ( 4 0 ) . W i t h many s e n s i t i z e r s , the r a t e of p h o t o o x i d a t i o n of guanine and i t s d e r i v a t i v e s i n c r e a s e s w i t h i n c r e a s i n g pH i n a manner i n d i c a t i n g t h a t the a n i o n i c forms of these compounds, as w i t h some amino a c i d s , are the most s e n s i t i v e t o a t t a c k . The p h o t o o x i d a t i o n r a t e s of thymine and u r a c i l a l s o i n c r e a s e w i t h pH (5)· R e l a t i v e l y l i t t l e i s known about the m e c h a n i s t i c d e t a i l s of the p h o t o o x i d a t i o n of p u r i n e s and p y r i m i d l n e s . T y p i c a l l y , complex a r r a y s of r e a c t i o n p r o d u c t s are formed t h a t are p r o b a b l y d e r i v e d from u n s t a b l e d i o x e t a n e s , endoperoxides or h y d r o p e r o x i d e s ( 7 , 2 1 ) . The methylene blue s e n s i t i z e d p h o t o o x i d a t i o n of guanosine r e s u l t s i n the u l t i m a t e p r o d u c t i o n of g u a n i d i n e , r i b o s e , r i b o s y l u r e a and u r e a f

f

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f

,

f

( 4 1 ) . The Type I o x i d a t i o n p r o d u c t s of 3 , 5 - d i - 0 - a c e t y l - 2 deoxyguanosine i n c l u d e two anomers o f 3 , 5 - d i - 0 - a c e t y l - 2 - d e o x y e r y t h r o p e n t o f u r a n o s e p l u s s e v e r a l u n i d e n t i f i e d compounds. The predominant s i n g l e t oxygen o x i d a t i o n p r o d u c t s o f t h i s guanosine d e r i v a t i v e are N-(3,5-di-0-acetyl-2-deoxy-erythropentofuranosyl)c y a n u r i c a c i d and 9 - ( 3 , 5 - d i - 0 - a c e t y l - 2 - d e o x y - e r y t h r o p e n t o f u r a n o s y l ) 4,8-dihydro-4-hydroxy-8-oxoguanine ( 3 7 ) . The primary p h o t o o x i d a t i o n product o f u r a c i l , as determined a t v e r y low temperatures, appears t o be a v e r y u n s t a b l e h y d r o p e r o x i d e ( 4 2 ) . 1

,

,

Photodynamic E f f e c t s on N u c l e i c A c i d s . N u c l e i c a c i d s a r e p h o t o o x i d i z e d on i l l u m i n a t i o n i n the presence of a number o f d i f f e r e n t k i n d s of p h o t o s e n s i t i z e r s ; i n e s s e n t i a l l y a l l c a s e s , photodynamic treatment o f n u c l e i c a c i d s and s y n t h e t i c p o l y n u c l e o t i d e s p r e f e r e n t i a l l y d e s t r o y s guanine r e s i d u e s ( 5 , 3 7 ) . For example, i t has been shown t h a t h e m a t o p o r p h y r i n , which does n o t intercalate into nuclei p h o t o a l t e r a t i o n of guanin subsequent treatment w i t h base r e s u l t s i n c h a i n breaks a t each guanine r e s i d u e . Methylene b l u e , which, l i k e o t h e r b a s i c dyes i n t e r c a l a t e s i n t o the n u c l e i c a c i d h e l i x , s p e c i f i c a l l y s e n s i t i z e s the p h o t o a l t e r a t i o n of guanine r e s i d u e s i n both s i n g l e - s t r a n d e d and d o u b l e - s t r a n d e d DNA. A l t h o u g h the hematoporphyrin r a d i c a l a n i o n i s produced i n good y i e l d on i l l u m i n a t i o n i n the presence o f DNA, i t does not i n t e r a c t w i t h the n u c l e i c a c i d . S i n g l e t oxygen appears t o be the r e a c t i v e s p e c i e s w i t h both hematoporphyrin and methylene b l u e (43) . I n c o n t r a s t , photodynamic treatment of DNA w i t h rose bengal (44) o r w i t h r i b o f l a v i n (45) generates s i n g l e - s t r a n d c h a i n b r e a k s , a p p a r e n t l y by i n t e r a c t i o n of the t r i p l e t s e n s i t i z e r s w i t h t h e n u c l e i c a c i d . S i n g l e t oxygen does not appear t o be i n v o l v e d . Superoxide and hydrogen p e r o x i d e have a l s o been suggested as being r e a c t a n t s i n the photodynamic d e g r a d a t i o n o f n u c l e i c a c i d s ( 4 6 ) . I n a d d i t i o n t o s t r a n d breakage, p h o t o d y n a m i c a l l y - t r e a t e d n u c l e i c a c i d s show c o n f o r m a t i o n a l a l t e r a t i o n s , s p e c t r a l s h i f t s , a decrease i n s o l u t i o n v i s c o s i t y and m e l t i n g temperature, and an i n c r e a s e d s u s c e p t i b i l i t y t o enzymatic d i g e s t i o n (5). Major changes i n b i o l o g i c a l a c t i v i t y a l s o o c c u r . F o r example, tobacco mosaic v i r u s RNA l o s e s i t s a b i l i t y t o i n f e c t tobacco p l a n t s , DNA t r a n s f o r m i n g p r i n c i p l e from b a c t e r i a i s d e s t r o y e d , and t r a n s f e r RNA i s i n a c t i v a t e d ; f u r t h e r , the messenger, template and t r a n s l a t i o n a l a c t i v i t i e s of n u c l e i c a c i d s a r e a l t e r e d (5,46,47). M u t a t i o n s can be produced i n b a c t e r i o p h a g e DNA by photodynamic treatment ( 4 8 ) . Photodynamic E f f e c t s on M i s c e l l a n e o u s B i o m o l e c u l e s . In addition to the major c a t e g o r i e s d e s c r i b e d above, a number of o t h e r k i n d s o f b i o m o l e c u l e s a r e s e n s i t i v e t o photodynamic a t t a c k (_1_). F o r example, ascorbic acid i s photooxidized with porphyrin s e n s i t i z e r s ; the a s c o r b a t e f r e e r a d i c a l i s produced i n the r e a c t i o n ( 4 9 ) . The p l a n t hormone, i n d o l e - 3 - a c e t i c a c i d , i s p h o t o o x i d i z e d u s i n g FMN; competing Type I and Type I I p r o c e s s e s a r e i n v o l v e d ( 5 0 ) . E o s i n Y and methylene b l u e s e n s i t i z e the p h o t o o x i d a t i o n o f a l p h a - k e t o g l u t a r i c a c i d i n a s i n g l e t oxygen mediated p r o c e s s ; s u c c i n i c a c i d and carbon d i o x i d e a r e produced i n the r e a c t i o n ( 5 1 ) . The i l l u m i n a t i o n o f p h y t o l i n the presence of rose bengal o r methylene b l u e g i v e s two

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a l l y l i c h y d r o p e r o x i d e s i n a r e a c t i o n i n v o l v i n g s i n g l e t oxygen (52)· Squalene i s p h o t o o x i d i z e d w i t h c h l o r p r o m a z i n e v i a a s i n g l e t oxygen r e a c t i o n w i t h the f o r m a t i o n of p e r o x i d i z e d p r o d u c t s ( 5 3 ) . The f l a v a n o l , q u e r c e t i n , i s s l o w l y p h o t o o x i d i z e d i n a s e l f s e n s i t i z e d p r o c e s s , as w e l l as i n a r e a c t i o n s e n s i t i z e d by r i b o f l a v i n . The r a t e of the f l a v i n e - s e n s i t i z e d p h o t o o x i d a t i o n i s i n c r e a s e d 1 0 - f o l d i n the presence of EDTA i n a r e a c t i o n i n h i b i t e d by superoxide dismutase, s u g g e s t i n g t h a t the process i s mediated, at l e a s t i n p a r t , by s u p e r o x i d e ( 5 4 ) . V i t a m i n Ε ( a l p h a - t o c o p h e r o l ) r e a c t s w i t h s i n g l e t oxygen produced by photodynamic s e n s i t i z e r s by a r a p i d p h y s i c a l quenching p r o c e s s , and by a s l o w e r , i r r e v e r s i b l e r e a c t i o n t h a t g i v e s two i s o m e r i c hydroperoxydienones ( 5 5 ) . The i l l u m i n a t i o n of v i t a m i n Ε i n the presence of hematoporphyrin d e r i v a t i v e l e a d s to the uptake of oxygen and the f o r m a t i o n of the v i t a m i n Ε chromanoxyl f r e e r a d i c a l ( 5 6 ) . Photodynamic treatment of v i t a m i n B12 w i t h methylene b l u e as s e n s i t i z e r r e s u l t s i n two photooxygenated p r o d u c t pigments and model m e l a n i n r a t e i s g r e a t l y enhance y benga appears to i n v o l v e s i n g l e t oxygen, at l e a s t i n p a r t . Rose bengal a l s o s e n s i t i z e s an i n c r e a s e i n the p h o t o p r o d u c t i o n of f r e e r a d i c a l s i n melanins (58)· B i o c h e m i s t r y of Photodynamic R e a c t i o n s

i n C e l l s and

Organelles

I n f o r m a t i o n o b t a i n e d on p h o t o s e n s i t i z e d r e a c t i o n s i n s o l u t i o n s t u d i e s cannot always be a p p l i e d d i r e c t l y to s t u d i e s w i t h c e l l s . T h i s i s because c e l l s and s u b c e l l u l a r s t r u c t u r e s are nonhomogeneous, w i t h r e g i o n s t h a t d i f f e r w i d e l y i n c h e m i c a l makeup. Thus c e l l s p r o v i d e a l a r g e range of microenvironments w i t h d i f f e r e n t p h y s i c o c h e m i c a l p r o p e r t i e s . As a r e s u l t , the p h o t o s e n s i t i z i n g p r o p e r t i e s of s e n s i t i z e r s can depend on t h e i r p a r t i c u l a r c e l l u l a r environment; s i m i l a r l y the r e a c t i o n s of s u b s t r a t e s may be d i f f e r e n t i n d i f f e r e n t r e g i o n s of the c e l l . Most types of b i o m o l e c u l e s i n i n t a c t c e l l s and i n i s o l a t e d c e l l u l a r o r g a n e l l e s show the same k i n d s of b i o c h e m i c a l changes on photodynamic treatment as observed i n s o l u t i o n ( 1 , 2 ) . For example, s u s c e p t i b l e amino a c i d r e s i d u e s i n c e l l membrane p r o t e i n s are p h o t o o x i d i z e d e f f i c i e n t l y ( 1 , 5 9 ) . T h i s r e s u l t s i n the photodynamic i n a c t i v a t i o n of a number of membrane a s s o c i a t e d enzymes i n c l u d i n g glyceraldehyde-3-phosphate dehydrogenase, ATPases, a c e t y l c h o l i n e s t e r a s e , e t c . Enzymes a s s o c i a t e d w i t h m i t o c h o n d r i a , such as s u c c i n i c dehydrogenase, ATPase, and a d e n y l a t e k i n a s e are a l s o i n a c t i v a t e d e f f i c i e n t l y w i t h some s e n s i t i z e r s ( 6 0 ) ; among o t h e r e f f e c t s , t h i s may r e s u l t i n decreased l e v e l s of ATP i n t r e a t e d c e l l s , which can i n t e r f e r e w i t h a number of d i f f e r e n t c e l l u l a r a c t i v i t i e s (61) · S o l u b l e enzymes i n the c y t o s o l of the c e l l are a l s o s e n s i t i v e . I l l u m i n a t i o n of p h o t o s e n s i t i z e d c e l l membranes, e.g., red blood c e l l g h o s t s , i n the presence of p o r p h y r i n s , g i v e s a s i n g l e t oxygen-mediatd p e r o x i d a t i o n of the membrane l i p i d s ; the r a t e of p e r o x i d a t i o n i s s i g n i f i c a n t l y i n c r e a s e d by low c o n c e n t r a t i o n s of a s c o r b a t e ( 6 2 ) . The photodynamic treatment of i s o l a t e d microsomes r e s u l t s i n the p e r o x i d a t i o n of the component l i p i d s and i n the i n a c t i v a t i o n of the mixed f u n c t i o n o x i d a s e system. I l l u m i n a t i o n of

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h e p a t i c microsomes from r a t s p r e t r e a t e d w i t h hematoporphyrin d e r i v a t i v e r e s u l t s i n a r a p i d d e s t r u c t i o n of cytochrome P-450 ( 6 3 ) . As i n s o l u t i o n , s e n s i t i z e d p h o t o c r o s s - l i n k i n g r e a c t i o n s o c c u r w i t h b i o m o l e c u l e s i n c e l l o r g a n e l l e s and i n i n t a c t c e l l s . A c t u a l l y , such r e a c t i o n s may be f a v o r e d i n c e l l s as compared to s o l u t i o n systems because of the h i g h l y o r d e r e d s t r u c t u r e of c e l l s ( 6 4 ) . DNA s t r a n d breaks occur i n murine f i b r o b l a s t c e l l s i l l u m i n a t e d i n the presence of hematoporphyrin d e r i v a t i v e ; t h i s a p p a r e n t l y r e s u l t s from the p h o t o o x i d a t i o n of guanine r e s i d u e s i n the n u c l e i c a c i d ( 6 5 ) . Summary We now understand the i n i t i a l r e a c t i o n s i n v o l v e d i n the b i o c h e m i s t r y of photodynamic a c t i o n r e a s o n a b l y w e l l , i . e . , the p r o d u c t i o n and p r o p e r t i e s of the e x c i t e d s t a t e s o f s e n s i t i z e r s and the i n t e r a c t i o n of t r i p l e t s e n s i t i z e r s w i t h ground s t a t e oxygen to g i v e s i n g l e t oxygen. The i n t e r a c t i o becoming c l e a r e r , but th b i o l o g i c a l s u b s t r a t e s v i a f r e e r a d i c a l p r o c e s s e s are more complex. Much remains to be l e a r n e d about the m e c h a n i s t i c o r g a n i c c h e m i s t r y of these r e a c t i o n s . F i n a l l y , a l t h o u g h p r o g r e s s i s being made, our u n d e r s t a n d i n g of p h o t o s e n s i t i z e d r e a c t i o n s at t h e c e l l u l a r and o r g a n i s m a l l e v e l s i s s t i l l v e r y i n c o m p l e t e . There i s much work yet to be done on the b i o c h e m i s t r y of photodynamic a c t i o n . Acknowledgments. The p r e p a r a t i o n of t h i s r e v i e w was supported i n p a r t by American Cancer S o c i e t y Grant #PDT-259A, by the V e t e r a n s A d m i n i s t r a t i o n M e d i c a l Research Program, and by the O f f i c e of N a v a l R e s e a r c h , C o n t r a c t No. N00014-88-K-0285. Literature Cited 1. 2. 3. 4. 5. 6. 7.

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9. 10. 11. 12.

Spikes, J.D. In Photoimmunology; Parrish, J . Α . ; Kripke, M.L.; Morison, W.L., Eds.; Plenum: New York, 1983; Chapter 2. Spikes, J.D. In The Science of Photomedicine; Regan, J . D . ; Parrish, J . Α . , Eds.; Plenum: New York, 1982; Chapter 5. Tappeiner, H. v. Ber. Deut. Chem. Ges. 1903, 36, 3035. Blum, H.F. Photodynamic Action and Diseases Caused by Light; Hafner Publishing Company: New York, 1964. Straight, R.C.; Spikes, J.D. In Singlet O ; Frimer, Α.Α., Ed.; CRC Press: Boca Raton, 1985; Vol IV, Chapter 2. Foote, C.S. In Free Radicals in Biology; Pryor, W.A., Ed.; Academic: New York, 1976; Vol II, Chapter 3. Foote, C.S. In Oxygen and Oxy-Radicals in Chemistry and Biology; Rodgers, M.A.J.; Powers, E . L . , Eds.; Academic: New York, 1981; p 425. Foote, C.S. In Porphyrin Localization and Treatment of Tumors; Doiron, D.R.; Gomer, C.J., Eds.; Alan R. Liss: New York, 1984; p 3. Laustriat, G. Biochimie 1986, 68, 771. Towers, G.H.N. Prog. Phytochem. 1980, 6, 1983. Andley, U.P.; Chakrabarti, B. Biochem. Biophys. Res. Commun. 1983, 115, 894. Terao, J.; Matsushita, S. Agric. Biol. Chem. 1981, 45, 601. 2

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6. SPIKES AND STRAIGHT 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

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Frimer, Α.Α.; Stephenson, L.M. In Singlet O ; Frimer, A . A . , Ed.; CRC Press: Boca Raton, 1985; Vol II, Chapter 3. Teng, J.I.; Smith, L . L . J. Am. Chem. Soc. 1973, 95, 4060. Jasiczak, J.; Smoczkiewica, M.A. Tetrahedron Lett. 1985, 26, 5221. Sedee, A.G.J.; Beijersbergen van Henegouwen, G.M.J.; Lusthof, K . J . ; Lodder, G. Biochem. Biophys. Res. Commun. 1984, 125, 675. Sedee, Α.; Vanhenegouwen, G.B. Arch. Pharm. 1985, 318, 111. Ando, W.; Takata, T. In Singlet O ; Frimer, Α.Α., Ed.; CRC Press: Boca Raton, 1985; Vol III, Chapter 1. Buettner, G.R. FEBS Lett. 1985, 177, 295. Gennari, G.; Cauzzo, G.; Jori, G. Photochem. Photobiol. 1974, 20, 497. Wasserman, H.W.; Lipshutz, B.H. In Singlet Oxygen; Wasserman, H.W.; Murray, R.W., Eds.; Academic Press: New York, 1979; Chapter 9. Sysak, P.K.; Foote 1977, 26, 19. Bowen, J.R.; Yang, S.F. Photochem. Photobiol. 2975, 21, 201. George, M.V.; Bhat, V. Chem. Rev. 1979, 79, 447. Nakagawa, M.; Yokoyama, Y.; Kato, S.; Hino, T. Tetrahedron 1985, 41, 2125. Ishimitsu, S.; Fujimoto, S.; Ohara, A. Chem. Pharm. Bull. 1985, 33, 1552. Rizzuto, F.R.; Spikes, J . D . ; Coker, G.D. Photobiochem. Photobiophys. 1986, 10, 149. Straight, R.C.; Spikes, J.D. Photochem. Photobiol. 1978, 27, 565. Jori, G.; Galiazzo, G.; Tamburro, A.M; Scoffone, E. J . Biol. Chem. 1970, 245, 3375. Jori, G. An. Acad. Bras. Cien. 1973. 45, 33. Rippa, M.; Pontremoli, S. Arch. Biochem. Biophys. 1969, 103, 112. Hopkins, T.R.; Spikes, J.D. Photochem. Photobiol. 1970, 12, 175. Brandt, J. Methods Enzymol. 1977, 46, 561. Verweij, H.; Dubbleman, T.M.A.R.; Van Steveninck, J . Biochim. Biophys. Acta. 1981, 647, 87. Helene, C. In Aging, Carcinogenesis and Radiation Biology; Smith, K . C . , Ed.; Plenum Press: New York, 1976; p 149. Hemmendorf, B.; Brandt, J.; Anderson, L.-O. Biochim. Biophys. Acta. 1981, 667, 15. Cadet, J.; Berger, M.; Decarroz, C.; Wagner, J.R.; Van Lier, J . E . ; Ginot, Y.M.; Vigny, P. Biochemie 1986, 68, 813. Jori, G.; Spikes, J.D. In Topics in Photomedicine; Smith, K.C., Ed.; Plenum Press: New York, 1984; p 183. Cadet, J.; Decarroz, C.; Voituriez, L . ; Gaboriau, F . ; Vigny, P. In Oxygen Radicals in Chemistry and Biology; Bors, W.; Saran, M.; Tait, D., eds.; Walter de Gruyter: Berlin, 1984; p 485. Ennever, J . F . ; Speck, W.T. Pediatr. Res. 1981, 15, 956. Matsuura, T.; Saito, I. General Heterocyclic Chem. 1976, 4, 456. 2

2

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42. 43.

Vickers, R.S.; Foote, S. Boll. Chim. Farm. 1970, 109, 599. Kawanishi, S.; Inoue, S.; Sano, S.; Alba, H. J . Biol. Chem. 1986, 261, 6090. 44. Peak, M.J.; Peak, J . G . ; Foote, C.S.; Krinsky, N.I. J . Photochem. 1984, 25, 309. 45. Korycka-Dahl, M.; Richardson, T. Biochim. Biophys. Acta. 1980, 610, 229. 46. Amagasa, J. Photochem. Photobiol. 1981, 33, 947. 47. Spikes, J.D.; MacKnight, M.L. In Photochemistry of Macromolecules; Reinisch, R . F . , Ed.; Plenum Press: New York, 1970; p 67. 48. Piette, J.; Calberg-Bacq, D.M.; Van de Vorst, A. Mol. Gen. Genet. 1978, 167, 95. 49. Buettner, G.R.; Need, M.J. Cancer Lett. 1985, 25, 297. 50. Miyoshi, N.; Fukuda, M.; Tomita, G. Photobiochem. Photobiophys. 1986, 11, 57. 51. Gandhi, P.; Dubey, 1985, 54, 37. 52. Mihara, S.; Tateba, H. J . Org. Chem. 1986, 51, 1142. 53. Fujita, H.; Matsuo, I.; Okazaki, M.; Yuoshino, K.; Ohkido, M. Dermatol. Res. 1986, 278, 224. 54. Takahama, U. Photochem. Photobiol. 1985, 42, 89. 55. Clough, R.L.; Yee, B.G.; Foote, C.S. J . Am. Chem. Soc. 1979, 101, 683. 56. Buettner, G.R. In Primary Photo-Processes in Biology and Medicine; Bensasson, R.V.; Jori, G.; Land, E.J.; Truscott, T . G . , Eds.; Plenum: New York, 1985; p 341. 57. Kraeutler, B.; Stepanek, R. Agnew. Chem. 1985, 97, 71. 58. Sarna, T.; Menon, I . Α . ; Sealy, R.C. Photochem. Photobiol. 1985, 42, 529. 59. Moan, J.; Vistnes, A . I . Photochem. Photobiol. 1986, 44, 15. 60. Fu, N.; Yeh, S.; Chang, C.; Zhao, X.; Chang, L . Adv. Exp. Med. Biol. 1985, 193, 161. 61. Hilf, R.; Murant, R.S.; Narayanan, U.; Gibson, S.L. Cancer Res. 1986, 46, 211. 62. Girotti, A.W.: Thomas, J . P . ; Jordan, J . E . Photochem. Photobiol. 1985, 41, 267. 63. Das, M.; Dixit, R.; Mukhtar, H.; Bickers, D.R. Cancer Res. 1985, 45, 608. 64. Jori, G.; Spikes, J.D. In Oxygen and Oxy-Radicals in Chemistry and Biology; Rodgers, M.A.J.; Powers, E . L . , Eds.; Academic Press: New York, 1981; p 441. 65. Dubbelman, T.M.A.R.; Boegheim, J . P . J . ; Van Steveninck, J . In Primary Photo-Processes in Biology and Medicine; Bensasson, R.V.; Jori, G.; Land, E.J.; Truscott, T . G . , Eds.; Plenum: New York, 1985; p 397. RECEIVED February 11, 1987

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 7

Photodynamic Modification of Excitable Cell Function John P. Pooler Department of Physiology, Emory University School of Medicine, Atlanta, GA 30322

Photodynamic treatment of electrically excitable cells from nerve and muscl dose-dependent perturbation in thei axons, particularl induced to fire action potentials. Voltage clamp analyses of photomodified lobster axons reveal a block of sodium channels and an inhibition of inactivation gating in unblocked channels. Potassium channels (delayed rectifiers) are less susceptible, but also become blocked and have a slowed activation. An unidentified ion leakage is created that is probably responsible for triggering the light-induced firing. The susceptibility to firing observed in vertebrate neuromuscular junctions suggests that insect neuromuscular junctions may be a likely target for light-activated pesticides.

The nervous system has been i m p l i c a t e d as a key t a r g e t o f l i g h t a c t i v a t e d p e s t i c i d e s suggested in p a r t by b e h a v i o r a l a b n o r m a l i t i e s i n d i c a t i v e o f l o s s o f neuromuscular c o n t r o l Although t h e r e i s no d i r e c t e v i d e n c e to s u p p o r t t h i s c o n t e n t i o n , i t i s known t h a t t h e nervous systems o f a l l organisms a r e h i g h l y s u s c e p t i b l e to p e r t u r b a t i o n by t o x i n s and p h a r m a c o l o g i c a l agents (2), and nerve c e l l s from n o n - i n s e c t s p e c i e s have been shown t o be e a s i l y photomodified (3-9). The purpose o f t h i s c h a p t e r i s to d e s c r i b e e x i s t i n g s t u d i e s on photodynamic m o d i f i c a t i o n o f e x c i t a b l e c e l l s and p o i n t out how these f i n d i n g s might a p p l y to the a c t i o n s o f l i g h t activated pesticides. Nervous systems c a r r y out the f u n c t i o n o f s i g n a l i n g , from one r e g i o n o f a c e l l to another and from one end o f an organism to t h e other. A l l nervous systems a r e composed o f s i m i l a r elements and behave a c c o r d i n g to uniform p r i n c i p l e s at t h e c e l l u l a r l e v e l , i n much the same way t h a t complex e l e c t r o n i c d e v i c e s o f d i v e r s e f u n c t i o n are composed o f i d e n t i c a l elementary components ( 1 0 J . S t u d e n t s o f n e u r o b i o l o g y have u s u a l l y chosen s p e c i e s f o r study more

0097-6156/87/0339-0109$06.00/0 © 1987 American Chemical Society

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f o r c o n v e n i e n c e , such as ready a v a i l a b i l i t y and ease o f d i s s e c t i o n , than f o r any u n i q u e n e s s i n p r i n c i p l e o f o p e r a t i o n . Although s t u d i e s on the photodynamic m o d i f i c a t i o n s o f nervous systems have been c a r r i e d out on organisms o t h e r than t h o s e t h a t are t a r g e t s f o r l i g h t - a c t i v a t e d p e s t i c i d e s , one may c a u t i o u s l y e x t r a p o l a t e a c r o s s s p e c i e s and g e n e r a l i z e r e s u l t s from one s p e c i e s to a l l o t h e r s . Thus i t i s v e r y l i k e l y t h a t mechanisms s t u d i e d on t r a d i t i o n a l p r e p a r a t i o n s such as s q u i d n e r v e s and f r o g muscles a p p l y e q u a l l y to insect excitable c e l l s . The common elemental p r o c e s s e s found in a l l nervous systems i n c l u d e c o n d u c t i o n o f nerve i m p u l s e s ; the s t i m u l a t i o n or i n h i b i t i o n o f one c e l l by another at s y n a p t i c c o n t i g u i t i e s ; and spontaneous e x c i t a t i o n in a p p r o p r i a t e pacemaker and s e n s o r y t r a n s d u c e r c e l l s . Many o f t h e s e same events o c c u r in muscle t i s s u e as w e l l . D i s t u r b a n c e in any o f t h e s e p r o c e s s e s by photodynamic means i s potentially lethal. Historical

Background

Nerve-muscle p r e p a r a t i o n s . The f i r s t s u b s t a n t i a l i n v e s t i g a t i o n o f photodynamic m o d i f i c a t i o n o f e x c i t a b l e c e l l s was a s e r i e s o f s t u d i e s by L i p p a y i n i t i a t e d in the l a t e 1920s on n e r v e - m u s c l e p r e p a r a t i o n s from f r o g s , i . e . , i s o l a t e d f r o g s k e l e t a l muscles w i t h the d i s t a l p o r t i o n s o f t h e motor nerves t h a t i n n e r v a t e them s t i l l a t t a c h e d (1113). He found t h a t i l l u m i n a t i o n o f muscle s e n s i t i z e d w i t h any o f s e v e r a l xanthene s e n s i t i z e r s o r hematoporphyrin l e d to c o n t r a c t i o n s . Some c o n t r a c t i o n s were slow and s u s t a i n e d ; o t h e r s were r a p i d and spasm-like. L i p p a y proposed t h a t the spasms were t r i g g e r e d by l i g h t - i n d u c e d f i r i n g in the motoneurons. Research in subsequent y e a r s has amply c o n f i r m e d L i p p a y ' s h y p o t h e s i s ( 1 4 - 1 7 ) . Recordings i n motoneurons show t h a t a c t i o n p o t e n t i a l s o r i g i n a t e in the d i s t a l p o r t i o n s o f nerve near the muscle and t r a v e l a n t i d r o m i c a l l y away from the m u s c l e . They a l s o a c t i v a t e the neuromuscular s y n a p s e , triggering contraction. I f r e v e r s i b l e neuromuscular b l o c k i n g agents are a d d e d , the l i g h t - i n d u c e d c o n t r a c t i l e spasms can be b l o c k e d reversibly. In v e r t e b r a t e s neuromuscular t r a n s m i s s i o n i s a c c o m p l i s h e d by r e l e a s e o f a c e t y l c h o l i n e , s t o r e d in small v e s i c l e s w i t h i n the p r e s y n a p t i c nerve t e r m i n a l . A p r e s y n a p t i c a c t i o n p o t e n t i a l causes the emptying o f the c o n t e n t s o f many such v e s i c l e s i n t o the narrow space between the nerve membrane and muscle membrane, the a c e t y l c h o l i n e then a c t i n g to d e p o l a r i z e the muscle membrane. Normally the a c e t y l c h o l i n e i s r a p i d l y degraded immediately f o l l o w i n g the nerve impulse by a c e t y l c h o l i n e s t e r a s e p r e s e n t in h i g h c o n c e n t r a t i o n in the c l e f t a r e a . An a d d i t i o n a l f i n d i n g on s e n s i t i z e d n e r v e - m u s c l e p r e p a r a t i o n s from f r o g s i s a l i g h t - i n d u c e d d e c l i n e in the a c e t y l c h o l i n e s t e r a s e a c t i v i t y ( 1 5 ) . With a c e t y l c h o l i n e h y d r o l y z e d more s l o w l y , a l i n g e r i n g d e p o l a r i z i n g a c t i o n on the muscle c e l l o c c u r s . S i n g l e nerve a c t i o n p o t e n t i a l s , t h a t n o r m a l l y t r i g g e r s i n g l e muscle a c t i o n p o t e n t i a l s and subsequent c o n t r a c t i l e t w i t c h e s , now t r i g g e r m u l t i p l e a c t i o n p o t e n t i a l s and twitches. Thus the s t i m u l a t i n g a c t i o n o f nerve on muscle f o r both l i g h t - i n d u c e d and e l e c t r i c a l l y s t i m u l a t e d f i r i n g i s a m p l i f i e d . In i n s e c t s the neuromuscular t r a n s m i t t e r i s L - g l u t a m a t e , and i t s

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i n a c t i v a t i o n i s m a i n l y by uptake i n t o g l i a l c e l l s r a t h e r than by enzymatic d e g r a d a t i o n ( 1 8 J . Whether i n s e c t neuromuscular j u n c t i o n s a r e as s e n s i t i v e to photodynamic p e r t u r b a t i o n as t h o s e in f r o g s i s unknown. A d i r e c t photodynamic a c t i o n on muscle membrane, not i n v o l v i n g neuromuscular t r a n s m i s s i o n , has a l s o been d e s c r i b e d ( 1 9 J . The muscle s l o w l y d e p o l a r i z e s and may break i n t o spontaneous f i r i n g , t h e r e b y t r i g g e r i n g c o n t r a c t i o n w i t h o u t any nerve a c t i v i t y . The muscle a c t i o n p o t e n t i a l s a l s o become i n c r e a s e d i n d u r a t i o n . A second d i r e c t a c t i o n on muscle i s the slow c o n t r a c t u r e o r i g i n a l l y d e s c r i b e d by L i p p a y , and c o n f i r m e d by o t h e r s ( H ) . It o c c u r s even w i t h neuromuscular t r a n s m i s s i o n blocked and the muscle made i n e x c i t a b l e by d e p o l a r i z a t i o n w i t h e l e v a t e d e x t r a c e l l u l a r p o t a s s i u m . The b a s i s f o r the slow 1 i g h t - i n d u c e d c o n t r a c t u r e i s u n r e s o l v e d at present. These photodynamic s t u d i e s on n e r v e - m u s c l e p r e p a r a t i o n s were c a r r i e d out over a spa t e c h n i c a l advances o r o r e v e a l e d by these t e c h n i q u e s . While the f i n d i n g s o f a l l i n v e s t i g a t o r s a r e not i n t o t a l harmony, most agree t h a t the most s e n s i t i v e t a r g e t i s some r e g i o n o f the nerve t e r m i n a l . The l i g h t doses r e q u i r e d to cause major d i r e c t muscle e f f e c t s a r e l a r g e r than t h o s e needed to evoke l i g h t - i n d u c e d f i r i n g i n the motoneurons. All o f the r e p o r t e d l i g h t - i n d u c e d e f f e c t s are p h o t o d y n a m i c - - i . e . , r e q u i r e o x y g e n - - a n d seem to be independent o f the p a r t i c u l a r s e n s i t i z e r used. Minor c o n f l i c t s between r e s u l t s i n d i f f e r e n t s t u d i e s may r e f l e c t d i f f e r e n c e s in the d i s t r i b u t i o n o f s e n s i t i z e r w i t h i n the complex anatomy o f the muscle at the time o f illumination. G i a n t axons. Almost a l l i n v e s t i g a t i o n s on photodynamic m o d i f i c a t i o n o f the nervous system have been c a r r i e d out on s i n g l e g i a n t axons i s o l a t e d from marine animals such as s q u i d , c u t t l e f i s h , and l o b s t e r . Most o f the work p r i o r to 1970 was performed by C h a l a z o n i t i s and coworkers i n France and by Lyudkovskaya and coworkers in the USSR (3-8). The axon r e g i o n o f a nerve c e l l i s r e l a t i v e l y " s i m p l e " in t h a t i t s s i g n a l i n g f u n c t i o n i s l i m i t e d to c o n d u c t i n g i m p u l s e s , i n i t i a t e d at one end or i n o t h e r c e l l r e g i o n s , along i t s l e n g t h to s y n a p t i c t e r m i n a l s at the o t h e r end. However, d e s p i t e a s t e r e o t y p e d normal f u n c t i o n , axons are c a p a b l e o f r a t h e r complex b e h a v i o r . The e l e c t r o p h y s i o l o g y o f axons can p r e s e n t l y be s t u d i e d at s e v e r a l l e v e l s o f s o p h i s t i c a t i o n , from s i m p l e e x t r a c e l l u l a r r e c o r d i n g f o r d e t e c t i o n o f a c t i o n c u r r e n t s a s s o c i a t e d w i t h the passage o f nerve i m p u l s e s , to " p a t c h clamp" methods f o r o b s e r v i n g the b e h a v i o r o f i n d i v i d u a l macromolecular ion c h a n n e l s . The s t u d i e s o f C h a l a z o n i t i s , L y u d k o v s k a y a , and a few o t h e r s who preceded them many decades ago employed e i t h e r e x t r a c e l l u l a r r e c o r d i n g or s i m p l e i n t r a c e l l u l a r r e c o r d i n g w i t h a p i p e t e l e c t r o d e i n s e r t e d i n t o the axon. These methods permit d e t e c t i o n o f f i r i n g p a t t e r n s and s u b t h r e s h o l d membrane d e p o l a r i z a t i o n , but do not a l l o w one t o d e s c r i b e 1 i g h t - i n d u c e d changes i n membrane p e r m e a b i l i t y . Use o f the v o l t a g e clamp method, to be d i s c u s s e d f u r t h e r o n , has r e v e a l e d how i o n i c p e r m e a b i l i t i e s are p h o t o d y n a m i c a l l y changed.

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C h a l a z o n i t i s and Lyudkovskaya both demonstrated t h a t s e n s i t i z e r - t r e a t e d g i a n t axons would commence r e p e t i t i v e f i r i n g d u r i n g i l l u m i n a t i o n , preceded by a l a t e n t p e r i o d d u r i n g which a slow depolarization occurred. The f i r i n g c o n t i n u e d a f t e r c e s s a t i o n o f i l l u m i n a t i o n f o r v a r y i n g p e r i o d s o f t i m e , but then s t o p p e d . Sometimes the membrane p o t e n t i a l would jump to a s t e a d y d e p o l a r i z e d l e v e l d u r i n g i l l u m i n a t i o n f o r s e v e r a l seconds and then r e t u r n to a v a l u e near the normal r e s t i n g p o t e n t i a l . In a s e p a r a t e study o f photodynamic m o d i f i c a t i o n o f g i a n t a x o n s , Lyudkovskaya d e s c r i b e d rather d i f f e r e n t r e s u l t s (8). In t h i s c a s e no l i g h t - i n d u c e d f i r i n g was r e p o r t e d . I n s t e a d Lyudkovskaya found major p e r t u r b a t i o n s i n the shape o f e l e c t r i c a l l y s t i m u l a t e d a c t i o n p o t e n t i a l s . They d e v e l o p e d a l o n g p l a t e a u between the r i s i n g and f a l l i n g p h a s e s , and the d u r a t i o n i n c r e a s e d from a t y p i c a l 1 o r 2 ms up to hundreds o f milliseconds. Both C h a l a z o n i t i s and Lyudkovskaya d e s c r i b e d the l i g h t - i n d u c e d f i r i n g as r e v e r s i b l e , w h i l e the p r o l o n g a t i o n o f e l e c t r i c a l l y stimulated actio irreversible. In 1968 P o o l e r i n i t i a t e d an i n v e s t i g a t i o n o f photodynamic m o d i f i c a t i o n o f g i a n t axons from l o b s t e r s ( 2 0 ) . He found an i r r e v e r s i b l e prolongation of e l e c t r i c a l l y stimulated action p o t e n t i a l s , as d i d L y u d k o v s k a y a , but no l i g h t - i n d u c e d f i r i n g . In a subsequent c l a r i f y i n g study d e s i g n e d to r e c o n c i l e the c o n f l i c t s i n r e p o r t e d f i n d i n g s , he found t h a t l i g h t - i n d u c e d f i r i n g c o u l d be i n i t i a t e d i n l o b s t e r g i a n t axons i f the b a t h i n g s o l u t i o n was f r e e o f calcium (21). C h a l a z o n i t i s and Lyudkovskaya had used such a c o n d i t i o n in t h e i r s t u d i e s d e s c r i b i n g 1 i g h t - i n d u c e d f i r i n g . Nerve c e l l s become h y p e r e x c i t a b l e i n s o l u t i o n s d e v o i d o f c a l c i u m , but a l s o d e t e r i o r a t e s l o w l y i f a c a l c i u m - f r e e c o n d i t i o n i s m a i n t a i n e d (22!). Thus g i a n t axons may be p h o t o d y n a m i c a l l y induced i n t o f i r i n g in conditions of low-calcium h y p e r e x c i t a b i l i t y . The f i r i n g i s a p p a r e n t l y r e v e r s i b l e , in t h a t f i r i n g c e a s e s at some p o i n t f o l l o w i n g t e r m i n a t i o n o f i l l u m i n a t i o n and may u s u a l l y be r e s t a r t e d by another dose o f l i g h t . The r e v e r s i b i l i t y i s a p p a r e n t , but not r e a l , because the c e s s a t i o n o f f i r i n g i s due in p a r t to d e t e r i o r a t i o n (and o t h e r f a c t o r s t o be d e s c r i b e d l a t e r ) r a t h e r than to removal o f t h e p h o t o m o d i f i c a t i o n t h a t i n i t i a t e s the f i r i n g . V o l t a g e Clamp A n a l y s i s o f Membrane Channel F u n c t i o n T e c h n i c a l background. Much o f our p r e s e n t u n d e r s t a n d i n g o f e x c i t a b l e c e l l f u n c t i o n stems from the s t u d i e s o f H o d g k i n , Huxley and K a t z , who a p p l i e d the v o l t a g e clamp t e c h n i q u e to g i a n t axons from s q u i d ( 2 3 J . When under v o l t a g e clamp the membrane p o t e n t i a l in a r e s t r i c t e d small area o f membrane i s c o n t r o l l e d by e l e c t r o n i c feedback. U s u a l l y the membrane p o t e n t i a l i s held a t a n e g a t i v e l e v e l from which a sequence o f s t e p d e p o l a r i z a t i o n s a r e a p p l i e d . At each v a l u e o f p o t e n t i a l to which the membrane i s c l a m p e d , the e l e c t r o c h e m i c a l d r i v i n g f o r c e f o r ion f l u x i s f i x e d . T h e r e f o r e the time c o u r s e o f c u r r e n t f l o w by any ion s p e c i e s r e f l e c t s the time c o u r s e o f membrane p e r m e a b i l i t y to t h a t i o n , u s u a l l y e x p r e s s e d in terms o f c o n d u c t a n c e (see E q u a t i o n 1) ( 2 4 ) . The t o t a l c u r r e n t t h r o u g h an axon membrane may be r e s o l v e c F i n t o t h r e e main components: sodium c u r r e n t , p o t a s s i u m c u r r e n t , and a n o n - s p e c i f i c leakage

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current. C u r r e n t s are r e l a t e d to membrane conductance and d r i v i n g f o r c e s as expressed in E q u a t i o n 1, where V i s t h e membrane p o t e n t i a l , Ε i s the r e v e r s a l p o t e n t i a l o f the a p p r o p r i a t e i o n , and g i s the c o n d u c t a n c e . I

= g (V - E)

(1)

The n o n - s p e c i f i c leakage conductance i s c o n s i d e r e d to be c o n s t a n t i n a normal membrane, w h i l e sodium and potassium c o n d u c t a n c e s a r e c l e a r l y v o l t a g e - and t i m e - d e p e n d e n t . T h e i r r e s p o n s e to a s t i m u l u s u n d e r l i e s the c h a r a c t e r i s t i c nerve impulse when the membrane p o t e n t i a l i s not c o n t r o l l e d by e l e c t r o n i c f e e d b a c k . I n v e s t i g a t i o n subsequent to the c l a s s i c s t u d i e s by H o d g k i n , Huxley and K a t z demonstrates t h a t the i o n i c conductance in a g i v e n f i n i t e r e g i o n o f membrane i s the sum o f the conductance o f a l l the i n d i v i d u a l i o n c h a n n e l s i n t h a t membrane. Each channel i s a polypeptide containing a region of r e s t r i c t i o n , givin c h a n n e l s a l s o c o n t a i n one o r more s e p a r a t e "gate regions that o c c l u d e the pore under some c o n d i t i o n s and open to a n o n - o c c l u d i n g c o n f o r m a t i o n under o t h e r c o n d i t i o n s . In t y p i c a l v o l t a g e clamp experiments the membrane r e g i o n under study c o n t a i n s up to a m i l l i o n channels. Ion conductances change smoothly over time i n r e s p o n s e to a s t e p change in p o t e n t i a l . T h i s smooth change r e p r e s e n t s the ensemble average o f many i n d i v i d u a l c h a n n e l s , each b e i n g e i t h e r open or c l o s e d . Thus the time c o u r s e o f the ensemble average r e f l e c t s the time c o u r s e o f p r o b a b i l i t y t h a t a g i v e n channel type i s o p e n . Sodium c h a n n e l s . When an axon membrane i s d e p o l a r i z e d , many sodium c h a n n e l s open ( a c t i v a t e ) but then c l o s e ( i n a c t i v a t e ) w i t h a somewhat slower time c o u r s e . Most i n v e s t i g a t o r s f e e l t h a t a c t i v a t i o n i s the opening o f a gate t h a t i s c l o s e d when the membrane i s p o l a r i z e d , and i n a c t i v a t i o n i s the c l o s i n g o f a p h y s i c a l l y s e p a r a t e gate t h a t i s n o r m a l l y open when the membrane i s p o l a r i z e d . When the membrane i s r e p o l a r i z e d t h e r e i s a r e v e r s a l o f the events t h a t o c c u r d u r i n g depolarization. The a c t i v a t i o n g a t e c l o s e s and the i n a c t i v a t i o n gate opens. Photodynamic M o d i f i c a t i o n

o f L o b s t e r G i a n t Axons

Block o f sodium c h a n n e l s . Photodynamic p e r t u r b a t i o n o f sodium channel f u n c t i o n can be expressed in terms o f changes i n g a t i n g v a r i a b l e s and maximum c o n d u c t a n c e . V o l t a g e clamp a n a l y s i s o f p h o t o m o d i f i e d l o b s t e r g i a n t axons r e v e a l s a d e c r e a s e in the maximum sodium c o n d u c t a n c e . During i l l u m i n a t i o n a t a c o n s t a n t dose r a t e t h e d e c r e a s e f o l l o w s a s i m p l e s u r v i v a l c u r v e , i . e . , an e x p o n e n t i a l time c o u r s e toward a zero asymptote ( 2 6 ) . The s i m p l e s t i n t e r p r e t a t i o n of t h i s b e h a v i o r i s t h a t i n c r e a s i n g numbers o f i n d i v i d u a l c h a n n e l s become t o t a l l y b l o c k e d . At l a r g e d o s e s e s s e n t i a l l y a l l c h a n n e l s a r e b l o c k e d and no measurable sodium c u r r e n t flows d u r i n g a depolarization. The r a t e c o n s t a n t f o r the development o f b l o c k d u r i n g i l l u m i n a t i o n v a r i e s l i n e a r l y w i t h dose r a t e and depends h e a v i l y on the s p e c i e s o f s e n s i t i z e r . When d i f f e r e n t s e n s i t i z e r s are compared under c o n d i t i o n s o f equal absorbed dose r a t e s , the r a t e

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o f channel block becomes a u s e f u l assay t o compare t h e i r p o t e n c y . For example, w i t h i n the f l u o r e s c e i n f a m i l y o f s e n s i t i z e r s r o s e bengal i s by f a r the most potent ( 2 7 ) . P e r t u r b a t i o n o f sodium channel g a t i n g . I l l u m i n a t i o n a l s o causes a complex d i s t u r b a n c e o f the i n a c t i v a t i o n gate i n n o n - b l o c k e d c h a n n e l s , l e a d i n g to a prolonged flow o f sodium c u r r e n t i n r e s p o n s e to d e p o l a r i z a t i o n ( 2 6 ) . F o l l o w i n g any f i n i t e l i g h t d o s e , t h e r e f o r e , t h e r e a r e t h r e e s u b p o p u l a t i o n s o f sodium c h a n n e l s : normal, b l o c k e d , and unblocked w i t h m o d i f i e d i n a c t i v a t i o n g a t i n g . The r e l a t i v e numbers o f t h e s e s u b p o p u l a t i o n s a r e shown s c h e m a t i c a l l y i n F i g u r e 1 as a f u n c t i o n o f l i g h t d o s e . Note t h a t the number with p e r t u r b e d i n a c t i v a t i o n g a t i n g r i s e s w i t h l i g h t dose up t o a maximum but then f a l l s at high doses because t h e m a j o r i t y o f t h e c h a n n e l s become blocked. D i s t u r b a n c e o f sodium channel i n a c t i v a t i o n r e v e a l s i t s e l f i n the k i n e t i c s o f i n a c t i v a t i o dose s u f f i c i e n t to b l o c which unblocked open c h a n n e l s i n a c t i v a t e d u r i n g a d e p o l a r i z a t i o n d e c r e a s e s by r o u g h l y 50% ( 2 6 ) . At the same t i m e , the s t e a d y - s t a t e i n a c t i v a t i o n versus voltage r e l a t i o n i s d i s t o r t e d . The s t e a d y - s t a t e v o l t a g e dependence f o r a c t i v a t i o n and i n a c t i v a t i o n g a t i n g may be expressed in terms o f the g a t i n g parameters m and h o f the HodgkinHuxley model a c c o r d i n g to E q u a t i o n s 2 and 3 , where V i s membrane p o t e n t i a l , V and V. a r e the membrane p o t e n t i a l s a t which the g a t i n g parameters are at h a l f maximum, and t h e k ' s i n d i c a t e t h e s t e e p n e s s o f the v o l t a g e dependence: m

œ

= 1/(1

+

exp((V - V J / k J )

(2)

h

e

= 1/(1

+

exp((V - V ) / k ) )

(3)

E q u a t i o n 3 may be r e v i s e d as i n E q u a t i o n 4: h

w

h

h

to i n c l u d e a n o n - i n a c t i v a t e d

= (1 - f ) / ( l

•

exp((V - V ) / k ) ) h

h

fraction,

+ f

f,

(4)

New experiments on l o b s t e r axons show t h e development o f a f o o t i n the i n a c t i v a t i o n c u r v e such t h a t some c h a n n e l s f a i l to i n a c t i v a t e at a l l at p o t e n t i a l s near z e r o (see F i g u r e 2 ) . A s i m i l a r b e h a v i o r has been found on squid g i a n t axons ( 2 8 ) . P o s s i b l e photodynamic p e r t u r b a t i o n o f a c t i v a t i o n g a t i n g i s small a t b e s t and below t h e r e s o l u t i o n o f t h e measurements. The a c t i v a t i o n parameter v a l u e s f o r p h o t o m o d i f i e d axons a r e not s i g n i f i c a n t l y d i f f e r e n t from t h o s e o f normal axons ( F i g u r e 2 ) . An e a r l i e r i n v e s t i g a t i o n r e v e a l e d no change in the k i n e t i c s o f a c t i v a t i o n e i t h e r (26). Thus i t appears t h a t p h o t o m o d i f i c a t i o n o f sodium channel g a t i n g i s l i m i t e d to the i n a c t i v a t i o n component. I n a c t i v a t i o n g a t i n g i s a complex p r o c e s s . Inactivation o c c u r r i n g as the c l o s u r e o f open c h a n n e l s d u r i n g a l a r g e d e p o l a r i z a t i o n may be d i f f e r e n t from i n a c t i v a t i o n o c c u r r i n g d u r i n g a small d e p o l a r i z a t i o n t h a t does not a c t i v a t e many c h a n n e l s ( 2 9 ) . T h i s second form o f i n a c t i v a t i o n i s c a l l e d c o n d i t i o n e d inactivation because i t i s measured by " c o n d i t i o n i n g " the membrane w i t h a small

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Photodynamic Modification of Excitable Cell Function

115

F i g u r e 1. Schematic i l l u s t r a t i o n o f photodynamic m o d i f i c a t i o n o f sodium c h a n n e l s showing d i v i s i o n o f t o t a l p o p u l a t i o n i n t o three f r a c t i o n s . B e f o r e i l l u m i n a t i o n a l l c h a n n e l s are n o r m a l , w h i l e a f t e r l a r g e l i g h t doses a l l are b l o c k e d . The unblocked c h a n n e l s w i t h m o d i f i e d i n a c t i v a t i o n r e a c h a maximum at an intermediate l i g h t dose.

Membrane

Potential

(mv)

F i g u r e 2. Steady s t a t e a c t i v a t i o n and i n a c t i v a t i o n v e r s u s membrane p o t e n t i a l f o r normal axons ( c o n t i n u o u s c u r v e s ) and p h o t o m o d i f i e d axons (dashed c u r v e s ) . The c u r v e s a r e p l o t s o f E q u a t i o n s 2 and 4 u s i n g mean parameter v a l u e s o b t a i n e d from 10 measurements each on normal axons and p h o t o m o d i f i e d axons s e n s i t i z e d w i t h 5 μΜ a c r i d i n e orange and i l l u m i n a t e d f o r a time s u f f i c i e n t to b l o c k 50% o f the sodium c o n d u c t a n c e .

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d e p o l a r i z a t i o n and then t e s t i n g f o r the amount o f i n a c t i v a t i o n d u r i n g a subsequent l a r g e d e p o l a r i z a t i o n . New experiments show the k i n e t i c s o f c o n d i t i o n e d i n a c t i v a t i o n on p h o t o m o d i f i e d l o b s t e r g i a n t axons to be u n a l t e r e d , and f u r t h e r m o r e , the k i n e t i c s o f r e c o v e r y from i n a c t i v a t i o n (the opening o f the i n a c t i v a t i o n g a t e upon repolarization) are not p e r t u r b e d e i t h e r ( F i g u r e 3 ) . Therefore, photodynamic m o d i f i c a t i o n o f sodium channel g a t i n g i s not o n l y l i m i t e d to i n a c t i v a t i o n , but to t h a t a s p e c t observed as the c l o s u r e o f open c h a n n e l s . P e r t u r b a t i o n o f potassium channel s and l e a k a g e . There are many k i n d s o f potassium c h a n n e l s i n e x c i t a b l e c e l l s . The predominant t y p e o f p o t a s s i u m channel i n axons i s c a l l e d a d e l a y e d rectifier. I t s g a t i n g has been modeled by the η parameter o f the Hodgkin-Huxley model ( 2 4 ) . More r e c e n t study o f d e l a y e d r e c t i f i e r s r e v e a l s the e x i s t e n c e o f a slow component i n the k i n e t i c s not d e a l t w i t h i n the Hodgkin and Huxley a n a l y s i s r e c t i f i e r (30). A descriptio delayed r e c t i f i e r s i s p r e s e n t l y incomplet i n t h e b e h a v i o r o f normal c h a n n e l s . N e v e r t h e l e s s , c e r t a i n f a c t s are apparent. F i r s t , p o t a s s i u m c h a n n e l s i n axons are b l o c k e d by photodynamic t r e a t m e n t . The s u s c e p t i b i l i t y , however, i s l e s s than t h a t o f sodium c h a n n e l s . I f the s u r v i v a l o f unblocked potassium and sodium c h a n n e l s i s compared under i d e n t i c a l r e a c t i o n c o n d i t i o n s , the r a t e o f potassium channel decay i s o n l y about 20% as g r e a t (21). S e c o n d , the a c t i v a t i o n g a t i n g o f potassium c h a n n e l s i s d i s t u r b e d ( f o r sodium c h a n n e l s i t i s n o t ) . The r a t e o f a c t i v a t i o n i s d e c r e a s e d and the slow component becomes more p r o m i n e n t . It i s not c l e a r whether t h i s r e p r e s e n t s a s e l e c t i v e b l o c k o f the f a s t component, making the o v e r a l l k i n e t i c s appear s l o w e r , o r whether the a c t i v a t i o n i s t r u l y slowed. In any event the o v e r a l l e f f e c t i s a d e c r e a s e in the l e v e l o f potassium conductance reached a t a g i v e n time f o l l o w i n g the s t a r t o f a s t e p d e p o l a r i z a t i o n . The m o d i f i c a t i o n t h a t p o t e n t i a l l y has the g r e a t e s t s i g n i f i c a n c e f o r axon f u n c t i o n — a n i n c r e a s e in l e a k a g e — h a s not been studied s y s t e m a t i c a l l y . T h i s i s because t h e v a s t m a j o r i t y o f v o l t a g e clamp s t u d i e s o f photodynamic m o d i f i c a t i o n have been c a r r i e d out on l o b s t e r g i a n t a x o n s , on which l e a k a g e cannot be measured f o r t e c h n i c a l reasons ( 3 1 ) . However, v o l t a g e clamp s t u d i e s performed on s q u i d g i a n t axons sïïôw a c o n s i s t e n t i n c r e a s e in leakage (unpublished). I n t e r p r e t a t i o n o f Nerve C e l l

Experiments

Two o f the key o b s e r v a t i o n s on nerve c e l l s seem to be in c o n f l i c t . Many s t u d i e s d e s c r i b e photodynamic m o d i f i c a t i o n as e x c i t a t o r y : nerve c e l l s a r e induced to f i r e d u r i n g l i g h t . Yet v o l t a g e clamp a n a l y s i s shows t h a t sodium c h a n n e l s become b l o c k e d by 1 i g h t — c l e a r l y an i n h i b i t o r y a c t i o n . ( L o c a l a n e s t h e t i c s work by b l o c k i n g sodium channels in axons.) The r e s o l u t i o n l i e s i n the f a c t t h a t axons have f a r more than the minimum d e n s i t y o f sodium c h a n n e l s r e q u i r e d to s u s t a i n a c t i o n p o t e n t i a l s ; thus events t h a t s t i m u l a t e an axon to f i r e may proceed even though sodium c h a n n e l s are b e i n g b l o c k e d simultaneously. ( I f they aVl_ become b l o c k e d , then f i r i n g w i l l

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

POOLER

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Membrane

Potential

< mv )

F i g u r e 3. Time c o n s t a n t f o r c o n d i t i o n e d i n a c t i v a t i o n and removal o f i n a c t i v a t i o n in normal axons ( t r i a n g l e s y m b o l s , c o n t i n u o u s l i n e s ) and p h o t o m o d i f i e d axons ( p l u s s y m b o l s , dashed lines). Each p o i n t i s the mean o f 11 or more o b s e r v a t i o n s . R e a c t i o n c o n d i t i o n s are the same as i n F i g u r e 2. The l a c k o f photodynamic e f f e c t stands in c o n t r a s t to the s l o w i n g o f i n a c t i v a t i o n when a s s e s s e d as the c l o s u r e o f open c h a n n e l s ( 2 6 ) .

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cease.) The l i g h t - i n d u c e d f i r i n g appears to be t r i g g e r e d by a 1 i g h t - i n d u c e d d e p o l a r i z a t i o n t h a t a c t s as a s t i m u l u s analogous to a sensory g e n e r a t o r p o t e n t i a l . The d e p o l a r i z a t i o n , i n t u r n , i s p r o b a b l y a r e s u l t o f the i n c r e a s e i n l e a k a g e . However, t h i s i n t e r p r e t a t i o n must be c o n s i d e r e d s p e c u l a t i v e u n t i l more d e t a i l i s l e a r n e d about t h e l i g h t - i n d u c e d l e a k a g e - - s p e c i f i c a l l y the ion species involved. In t h e o r y , d e p o l a r i z a t i o n can be brought about by a d e c r e a s e in p e r m e a b i l i t y to an ion s p e c i e s w i t h an e q u i l i b r i u m p o t e n t i a l more n e g a t i v e than the r e s t i n g p o t e n t i a l , such as potassium. K o h l i and B r y a n t ' s b r i e f r e p o r t on photodynamic d e p o l a r i z a t i o n o f s k e l e t a l muscle c e l l s (19) s t a t e s t h a t the e f f e c t was not seen when c h o l i n e was s u b s t i t u t e a f o r sodium, s u g g e s t i n g t h a t the d e p o l a r i z a t i o n i s caused by an anomalous r i s e in sodium permeability. L i g h t - i n d u c e d f i r i n g i s easy to produce on n e r v e - m u s c l e p r e p a r a t i o n s , but w i t h much more d i f f i c u l t y on g i a n t axons from marine a n i m a l s . Most, an d e s c r i b e d by Lyudkovskay p r e p a r a t i o n s made h y p e r e x c i t a b l e by l o w e r i n g the c a l c i u m c o n c e n t r a t i o n in the r e a c t i o n medium. Pooler described l i g h t induced f i r i n g on l o b s t e r axons i n c a l c i u m - f r e e m e d i a , but not w i t h high calcium c o n c e n t r a t i o n s p r e s e n t . On n e r v e - m u s c l e p r e p a r a t i o n s the t e r m i n a l p o r t i o n s o f the motoneurons e v i d e n t l y p o s s e s s a h i g h s u s c e p t i b i l i t y t o l i g h t - i n d u c e d f i r i n g even i n c o n d i t i o n s o f normal calcium. The r e a s o n s f o r t h i s a r e p r e s e n t l y unknown. The r e v e r s i b i l i t y o f l i g h t - i n d u c e d f i r i n g d e s c r i b e d by C h a l a z o n i t i s and Lyudkovskaya was shown to be an apparent r e v e r s i b i l i t y , due in p a r t to a slow d e t e r i o r a t i o n i n c a l c i u m - f r e e solutions. I f a nerve c e l l i s h y p e r e x c i t a b l e and t e e t e r i n g on the edge o f f i r i n g because o f low c a l c i u m a n d / o r a l i g h t - i n d u c e d d e p o l a r i z a t i o n , any small i n f l u e n c e can t r i g g e r f i r i n g or s t o p existing f i r i n g . Prolonged d e p o l a r i z a t i o n induces another form o f i n a c t i v a t i o n known as slow i n a c t i v a t i o n , not o r d i n a r i l y seen on the time s c a l e o f an a c t i o n p o t e n t i a l ( 2 5 J . A 1 ight-induced d e p o l a r i z a t i o n o r a l o n g t r a i n o f a c t i o n p o t e n t i a l s may cause slow i n a c t i v a t i o n , thus e f f e c t i v e l y r a i s i n g the f i r i n g t h r e s h o l d and h a l t i n g f i r i n g , but w i t h o u t r e v e r s i n g the p h o t o m o d i f i c a t i o n . M o d i f i c a t i o n o f g a t i n g in unblocked c h a n n e l s a l s o c o n t r i b u t e s to p e r t u r b a t i o n s in f i r i n g b e h a v i o r . The slowing o f i n a c t i v a t i o n k i n e t i c s and the f o o t in the i n a c t i v a t i o n c u r v e ( F i g u r e 2) both l e a d to a d i s t o r t i o n in shape and c o n s i d e r a b l e p r o l o n g a t i o n o f a c t i o n p o t e n t i a l d u r a t i o n , up to hundreds o f m i l l i s e c o n d s in some c a s e s . A v e r y l o n g a c t i o n p o t e n t i a l can i t s e l f s e r v e as a prolonged s t i m u l u s to nearby axon segments t h a t have r e c e i v e d a lower l i g h t d o s e . In most experiments o n l y a small segment i s i l l u m i n a t e d , t h u s p e r m i t t i n g e l e c t r o t o n i c i n t e r a c t i o n s between the m o d i f i e d r e g i o n and s u r r o u n d i n g unmodified segments. The f i n d i n g t h a t the c l o s u r e o f open c h a n n e l s i s s l o w e d , w h i l e the k i n e t i c s o f c o n d i t i o n e d i n a c t i v a t i o n remain n o r m a l , s u p p o r t s a p r e v i o u s l y s t a t e d c o n t e n t i o n (32) t h a t normal i n a c t i v a t i o n may o c c u r as two independent p r o c e s s e s — o n e t h a t o c c u r s o n l y f o l l o w i n g a c t i v a t i o n o f c h a n n e l s , and one t h a t i s independent o f a c t i v a t i o n .

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Chemical mechanisms. Channel f u n c t i o n i s v e r y e a s i l y p e r t u r b e d by a v a r i e t y o f p h a r m a c o l o g i c a l agents t h a t b l o c k a n d / o r modify g a t i n g . The open pore r e g i o n o f a channel t h a t d i s c r i m i n a t e s between d i f f e r e n t ion s p e c i e s (the s e l e c t i v i t y f i l t e r ) i s p r o b a b l y no more than a few Angstroms i n d i a m e t e r and i t w o u l d n ' t take a g r o s s s t r u c t u r a l a b n o r m a l i t y in t h i s p a r t o f a channel to b l o c k i o n movement ( 2 5 ) . The p h o t o c h e m i c a l mechanisms u n d e r l y i n g m o d i f i c a t i o n o f axon f u n c t i o n are not w e l l u n d e r s t o o d , however. V o l t a g e clamp experiments i n d i c a t e t h a t d i f f e r e n t s e n s i t i z e r s v a r y g r e a t l y in t h e i r p o t e n c y , but b r i n g about the same k i n d s o f a c t i o n s q u a l i t a t i v e l y , i m p l y i n g a common mode o f a c t i o n . (Lyudkovskaya d e s c r i b e d some s e n s i t i z e r - s p e c i f i c a c t i o n s i n n o n - v o l t a g e clamped a x o n s , but t h e s e have not been seen i n v o l t a g e clamp e x p e r i m e n t s . ) The major m o d i f i c a t i o n o f sodium c h a n n e l s — b l o c k and p e r t u r b a t i o n o f i n a c t i v a t i o n - - a r e p r o b a b l y independent p r o c e s s e s o c c u r r i n g in parallel. For a g i v e n degree o f channel b l o c k , however, the perturbation of i n a c t i v a t i o conditions. For example s e n s i t i z e r s , i s so e f f e c t i v e at s e n s i t i z i n g channe b l o c t h a t the r e l a t i v e l y low l i g h t doses r e q u i r e d to b l o c k 50% o f the c h a n n e l s may not p e r t u r b i n a c t i v a t i o n measurably (28, 3 3 ) . Thus s e n s i t i z e r a c c e s s i b i l i t y to a b l o c k i n g s i t e and an i n a c t i v a t i o n s i t e may v a r y somewhat from s e n s i t i z e r to s e n s i t i z e r . An i n c r e a s e in l e a k a g e c o u l d r e s u l t from m o d i f i c a t i o n o f e x i s t i n g c h a n n e l s , c r e a t i o n o f new pathways through o t h e r i n t e g r a l membrane p r o t e i n s or by a p e r t u r b a t i o n in t h e l i p i d b i l a y e r s t r u c t u r e . There are l i m i t e d d a t a p o i n t i n g to s i n g l e t oxygen as an intermediate. On l o b s t e r axons d e u t e r i u m o x i d e and a z i d e were a b l e to enhance and i n h i b i t channel b l o c k by about 50% e a c h , u s i n g r o s e bengal or e o s i n as s e n s i t i z e r s ( 3 4 ) . On s q u i d a x o n s , w i t h r e a g e n t s p e r f u s e d i n s i d e the c e l l , B - c a r o î ë n e e f f e c t i v e l y b l o c k e d s e n s i t i z a t i o n by methylene b l u e but not r o s e bengal (28). U n c e r t a i n t i e s in t h e d i s t r i b u t i o n o f r e a g e n t s w i t h i n tfie complex anatomy o f a c e l l u l a r system c l o u d s the i n t e r p r e t a t i o n o f t h e s e experiments. A p p l i c a t i o n to L i g h t - A c t i v a t e d P e s t i c i d e s S i n c e none o f the i n v e s t i g a t i o n on photodynamic m o d i f i c a t i o n o f e x c i t a b l e c e l l s has been performed on i n s e c t s p e c i e s , any e x t r a p o l a t i o n to l i g h t - a c t i v a t e d p e s t i c i d e s must o f n e c e s s i t y be very s p e c u l a t i v e . However, i f s e n s i t i z e r s i n c o n t a c t w i t h i n s e c t s can permeate t o e x c i t a b l e membranes ( i . e , they a r e not stopped by major d i f f u s i o n b a r r i e r s ) , i t seems v e r y l i k e l y t h a t the k i n d s o f m o d i f i c a t i o n s found in o t h e r l i f e forms would a l s o o c c u r in i n s e c t s . W i t h i n a whole organism t h e r e a r e many p h o t o m o d i f i a b l e s i t e s . So l o n g as the s e n s i t i z e r i s not photobleached then a l l s i t e s w i l l become m o d i f i e d at s u f f i c i e n t l y h i g h l i g h t d o s e s . To be o f p h o t o t o x i c i m p o r t a n c e , however, some must e x h i b i t p h o t o t o x i c p o t e n t i a l at r e l a t i v e l y low l i g h t d o s e s . W i t h i n a network o f nerve c e l l s an a r r a y o f elementary p r o c e s s e s can be p e r t u r b e d . At a g i v e n l i g h t dose some w i l l be f a r more m o d i f i e d than o t h e r s because o f d i f f e r e n t p r e - i l l u m i n a t i o n a s s o c i a t i o n s w i t h s e n s i t i z e r and d i f f e r e n t e f f e c t i v e quantum y i e l d s . The consequence o f a g i v e n

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modification w i l l also vary. (To use an analogy: A car can sustain a crushed bumper better than a broken spark plug.) At some point the accumulating modifications w i l l become manifestly toxic and survival of the organism threatened. Of the known photodynamic perturbations in excitable c e l l s , which ones have both a high s u s c e p t i b i l i t y to modification and a pivotal role in signaling? The obvious choice, block of sodium channels, i s probably not crucial because of the large excess number of channels r e l a t i v e to the minimum required to propagate action potentials. Only after many other perturbations have occurred i s i t l i k e l y that enough sodium channels would be blocked to halt propagation. Block of potassium channels i s also not l i k e l y to be crucial because of their r e l a t i v e l y low s e n s i t i v i t y to block. Interference with sodium channel inactivation may be more important because the prolongation of action poential duration that results from the interference decreases the maximum frequency of f i r i n g during a burst of action potentials. Even t h i s , however les important tha light induced f i r i n g at neuromuscula each muscle c e l l is a "slave and light-induced f i r i n g translates d i r e c t l y into muscle contraction. Extraneous muscle contraction interferes with locomotion. In turn, the disturbed locomotion translates into interference with feeding, escape from predators and reproduction. Whether this occurs in insects remains unknown, however. While neuromuscular transmission in insects has many mechanistic s i m i l a r i t i e s to that in vertebrates (35) the different transmitter substance employed, method of transmitter removal, and the difference in innervation pattern (18) makes this an open question. As noted in the introduction, one of the important elemental processes occurring in a l l nervous systems is spontaneous generation of excitation. The control of insect walking, in which muscles are alternately stimulated and i n h i b i t e d , i s thought to originate in a group of pacemaker c e l l s that undergo rhythmic o s c i l l a t i o n s in membrane potential (36). The frequency of o s c i l l a t i o n is continuously modulate? by synaptic input. Such l a b i l e c e l l s should be easily perturbed by photodynamic means. While no photodynamic studies have been carried out on insect pacemaker c e l l s , i t seems very l i k e l y that these c e l l s might be among the most susceptible to the action of light-activated pesticides.

Literature Cited 1. 2. 3. 4. 5. 6. 7.

Callaham, M. F . ; Lewis, L. Α.; Holloman, M. E.; Broome, J. E.; Heitz, J . R. Comp. Biochem. Physiol. 1975, 51C, 123-128. Narahashi, T. Physiol. Rev. 1974, 54, 813-889. Chalazonitis, N. These Sciences, Paris 1954, Ser. A, No. 2994, Order 3866, 1-116. Chalazonitis, N.; Chagneux, R. Bull. Inst. Oceanogr. (Monaco) 1961, 58(1223), 1-20. Chalazonitis, N. Photochem. Photobiol. 1964, 3, 534-559. Lyudkovskaya, R. G. Biofizika 1961, 6, 300-306. Lyudkovskaya, R. G.; Kayushin, L. P. Biofizika 1959, 4, 404412.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

7. POOLER 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.

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Lyudkovskaya, R. G.; Kayushin, L. P. Biofizika 1960, 5, 663670. Bostock, H. J . Physiol. 1982, 332, 57P-58P. Kuffler, S. W.; Nicholls, J . G . ; Martin, R. A. From Neuron to Brain, 2nd ed. Sinauer: Sunderland, MA, 1984. Lippay, F. Pflug. Arch. 1929, 222, 616-639. Lippay, F. Pflug. Arch. 1930, 224, 587-599. Lippay, F . ; Wechsler, L. Pflug. Arch. 1930, 224, 600-607. Rosenblum, W. I. J . Cell. Comp. Physiol. 1960, 55, 73-79. Burmistrov, Y. M.; Lyudkovskaya, R. G. Biofizika 1968, 13, 5565. Lyudkovskaya, R. G.; Pevzner, L. P. Biofizika 1964, 9, 580588. Sazonenko, Μ. Κ. Biofizika 1963, 8, 681-689. Usherwood, P. N. R.; Cull-Candy, S. G. In Insect Muscle; Usherwood, P. N. R., Ed.; Academic: London, 1975; pp. 207-280. Kohli, R. P.; Bryant Pooler, J . P. Biophys Pooler, J . P.; Oxford, G. S. J . Membr. Biol. 1973, 12, 339348. Adelman, W. J.; Adams, J. J . Gen. Physiol. 1959, 42, 655-664. Hodgkin, A. L.; Huxley, A. F . ; Katz, B. J . Physiol. 1952, 116, 424-448. Hodgkin, A. L.; Huxley, A. F. J . Physiol. 1952, 117, 500-544. Hille, B. Ionic Channels of Excitable Membranes. Sinauer: Sunderland, MA, 1984. Pooler, J . P. J. Gen. Physiol. 1972, 60, 367-387. Pooler, J . P.; Valenzeno, D. P. Photochem. Photobiol. 1979, 30, 491-498. Oxford, G. S.; Pooler, J . P.; Narahashi, T. J . Membr. Biol. 1977, 36, 159-173. Goldman, L. Q. Rev. Biophys. 1976, 9, 491-526. Dubois, J . M. Prog. Biophys. Molec. Biol. 1983, 42, 1-20. Pooler, J . P.; Valenzeno, D. P. Biophys. J . 1983, 44, 261-269. Oxford, G. S.; Pooler, J . P. J . Gen. Physiol. 1975, 66, 765779. Pooler, J . P.; Valenzeno, D. P. Photochem. Photobiol. 1978, 28, 219-226. Pooler, J . P.; Valenzeno, D. P. Photochem. Photobiol. 1979, 30, 581-584. Pichon, Y. In Insect Neurochemistry and Neurophysiology; Borkovec, A. B.; Kelly, T. J., Eds.; Plenum: New York, 1984; pp. 23-50. Shepherd, G. S. Neurobiology. Oxford: New York, 1983.

RECEIVED November 20, 1986

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 8

Physiological Effects of Photodynamic Action: Special Reference to Insects Joseph E. Weaver Division of Plant and Soil Sciences, West Virginia University, Morgantown, W V 26506

Results of studie i t s effect on th especially insects, With regards to insects, few studies on the photooxidative reactions were conducted until the early to mid-seventies. Since that time, there has been a renewed effort to determine how photodynamically active compounds affect insect physiology. This chapter is a review of findings on this phenomenon as it affects the organism at (1) the subcellular level, (2) the cellular level, (3) the systems level, and (4) aspects of photodynamic action as i t affects development and reproduction.

The p h y s i o l o g i c a l e f f e c t s o f photodynamic a c t i o n have been s t u d i e d i n a v a r i e t y o f o r g a n i s m s i n t h e l a s t e i g h t decades. However, o n l y f o u r o r i g i n a l papers f r o m 1900 t o 1970 r e p o r t e d o n i n s e c t s a s t h e e x p e r i m e n t a l e n t i t y . W h i l e some e a r l i e r s t u d i e s (1970-1975) designed t o evaluate the phototoxic e f f e c t s of photodynamically a c t i v e substances i n c l u d e d o b s e r v a t i o n s o n p h y s i o l o g i c a l e f f e c t s , t h i s s u b j e c t r e c e i v e d l i t t l e a t t e n t i o n from researchers u n t i l l a t e r i n t h e decade. B e g i n n i n g i n t h e m i d - s e v e n t i e s , and t o t h e p r e s e n t t i m e , s e v e r a l s t u d i e s have been conducted i n renewed e f f o r t s t o e l u c i d a t e t h e e f f e c t s t h a t a r e a s s o c i a t e d w i t h dyes e n s i t i z e d p h o t o o x i d a t i v e r e a c t i o n s i n i n s e c t s . These s t u d i e s have added g r e a t l y t o our knowledge o f how p h o t o d y n a m i c a l l y a c t i v e s u b s t a n c e s a f f e c t i n s e c t s (and o t h e r o r g a n i s m s ) f r o m t h e c e l l u l a r l e v e l t o the systems l e v e l . The phenomenon o f photodynamic a c t i o n has been t h e s u b j e c t o f s e v e r a l r e v i e w s . Some o f t h e works c i t e d i n those r e v i e w s have a l s o been c i t e d i n t h i s c h a p t e r where r e l e v a n t . These r e v i e w s a r e not a l l i n c l u s i v e and d e a l w i t h a s p e c t s o f photodynamism o t h e r t h a n p h y s i o l o g i c a l r a m i f i c a t i o n s i n l i v i n g organisms. The f o l l o w i n g a r e g e n e r a l r e v i e w s o f photodynamic e f f e c t s o n c e l l s a n d m u l t i c e l l u l a r o r g a n i s m s t h a t may be o f i n t e r e s t t o t h e r e a d e r : 0097-6156/87/0339-0122$06.00/0 © 1987 American Chemical Society

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Blum Q ) , E r r e r a (2), C l a r e (3), F o w l k s (4), S a n t a m a r i a (5-6). S a n t a m a r i a and P i n t o (J), S p i k e s and G h i r o n (8), Simon (£), S p i k e s and S t r a i g h t (10), Bourdon and S c h n u r i z e r (11), S p i k e s (12), S p i k e s and L i v i n g s t o n (13). P o o l e r and Valenzeno (14), and R o b i n s o n (15). The purpose of t h i s r e v i e w i s t o summarize and d i s c u s s f i n d i n g s on the p h y s i o l o g i c a l e f f e c t s of the photodynamic a c t i o n a t (1) t h e s u b c e l l u l a r l e v e l , ( I I ) the c e l l u l a r l e v e l , ( I I I ) t h e systems l e v e l , and (IV) a s p e c t s of the photodynamic a c t i o n as i t a f f e c t s development and r e p r o d u c t i o n i n i n s e c t s . E f f e c t s a t the S u b c e l l u l a r L e v e l Many s t u d i e s on v e r t e b r a t e s and i n v e r t e b r a t e s have shown t h a t "almost no c e l l p r o c e s s o r s t r u c t u r e i s immune f r o m ... photosens i t i z e d m o d i f i c a t i o n under the r i g h t c o n d i t i o n s " (14). S e n s i t i z e d m o l e c u l e s are a b l e t o o x i d i z including intermediates P o o l e r and Valenzeno (14) c i t e s e v e r a l examples of photo c h e m i c a l damage and m o d i f i c a t i o n s o c c u r r i n g t o i n t r a c e l l u l a r components by p h o t o s e n s i t i z i n g agents. Once p e r m e a b i l i t y of the c e l l membrane has been a l t e r e d , c e l l u l a r f u n c t i o n can be g r e a t l y modified. Cande e t a l . (16) r e p o r t e d t h a t l y s e d c e l l s of kangaroo r a t k i d n e y a r e permeable t o s m a l l m o l e c u l e s such as e r y t h r o s i n B. They observed t h a t h o l e s were p r e s e n t i n the plasma membrane and the m l t o c h r o n d i a were s w o l l e n and d i s t o r t e d ; o t h e r membrane-bound o r g a n e l l e s were not n o t i c e a b l y a l t e r e d . Haga and S p i k e s (17) a l s o reported s w e l l i n g of i s o l a t e d r a t l i v e r mitochondria s e n s i t i z e d w i t h e o s i n Y and methylene b l u e . On the b a s i s of c e r t a i n m e t a b o l i c measurements, they concluded t h a t the observed e f f e c t s o f t h e s e p h o t o t o x i n s s u g g e s t e d t h a t the s w e l l i n g i s caused by i n h i b i t i o n of enzymatic a c t i v i t i e s i n the e l e c t r o n t r a n s p o r t system and by the u n c o u p l i n g o f p h o s p h o r y l a t i o n f r o m r e s p i r a t i o n . H i l f e t a l . (18) have a l s o proposed t h a t m l t o c h r o n d i a may be a c r i t i c a l s u b c e l l u l a r s i t e of p h o t o m o d i f i c a t i o n . L o s s o f c e l l u l a r p o t a s s i u m has been r e p o r t e d and, i n t u r n , p r o t e i n s y n t h e s i s and c e l l membrane p o t e n t i a l s a r e a f f e c t e d . L y s o s o m a l damage by p h o t o s e n s i t i z e r s can r e s u l t i n secondary c e l l u l a r damage by a l t e r i n g the f i n e s t r u c t u r e o f m i t o c h o n d r i a and e n d o p l a s m i c r e t i c u l u m . U n s a t u r a t e d l i p i d s , n u c l e i c a c i d s , DNA, and RNA may be m o d i f i e d photo c h e m i c a l l y . P r o t e i n s and c e r t a i n o f t h e i r amino a c i d s i d e c h a i n s a r e s u s o e p t a b l e t o p h o t o s e n s i t i z e d attack. Palm (19) noted t h a t b a s i c dyes l i k e methylene b l u e and n e u t r a l r e d can produce g r a n u l e s i n the c y t o p l a s m of i n s e c t c e l l s w i t h n e u t r a l red g r a n u l e s o f t e n r e p r e s e n t a t i v e o f v a c u o l e s . Other w o r k e r s (see P o o l e r & Valenzeno (14)) have r e p o r t e d cytoplasmic v a c u o l i z a t i o n or blebs appearing i n photosensitized c e l l s . Only a l i m i t e d number o f s t u d i e s o f photodynamic a c t i o n i n i n s e c t s a t the s u b c e l l u l a r l e v e l have been conducted. C a r p e n t e r e t a l . (20) s t u d i e d the s y n e r g i s t i c e f f e c t o f f l u o r e s c e i n on r o s e bengal i n t h e presence of a p u r i f i e d enzyme. They showed t h a t f l u o r e s c e i n enhanced the photodynamic a c t i v i t y o f r o s e bengal i n

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the i n h i b i t i o n o f g l y c e r a l d e h y d e - 3 - p h o s p h a t e dehydrogenase of m o s q u i t o l a r v a e (Aedes t r i s e r i a t u s (Say)). Fondren and H e i t z (21) o b s e r v e d L T and t i s s u e l e v e l s o f dyes and suggested t h a t the f a c e f l y (Musca autumnal I s DeGeer) i s susceptable t o t o x i c I n t r a c e l l u l a r r e a c t i o n s of c e r t a i n s e n s i t i z e d xanthene dyes. Fondren e t a l . (22) conducted s i m i l a r s t u d i e s on t h e house f l y (Musca d o m e s t i c a L.) where " r e l a t i v e t o x i c i t i e s were d e s c r i b e d by means of r a t e c o n s t a n t s of p h o t o o x i d a t i o n c a l c u l a t e d f o r (6 xanthene dyes) w h i c h i n c l u d e d both t h e L T ^ Q and t h e t i s s u e dye l e v e l . " C a l l a h a m e t a l . (23) r e p o r t e d t h a t d y e - s e n s i t i z e d p h o t o o x i d a t i o n of the a c e t y l c h o l i n e s t e r a s e i n whole-head homogenates o f t h e i m p o r t e d f i r e ant ( S o l e n o p s l s r i c h t e r i ( F o r e l ) ) c o u l d be i n d u c e d i n the presence o f s e v e r a l xanthene dyes. L a c t i c dehydro genase and a c e t y l c h o l i n e s t e r a s e of the b o l l w e e v i l (Anthonomus g r a n d i s g r a n d i s Boheman) were i n a c t i v a t e d by d y e - s e n s i t i z e d p h o t o o x i d a t i o n mediated b s u b s t i t u t e d xanthene (24) There i s c o n s i d e r a b l a c t i v e compounds a r e mutagenic mutagenic c h e m i c a l s r e a c t w i t h the p r o t e i n p a r t of the gene m o l e c u l e r a t h e r t h a n w i t h the n u c l e i c a c i d (25). C l a r k (25) s t u d i e d the mutagenic a c t i v i t y of s e v e r a l dyes i n D r o s o p h i l a m e l a n o g a s t e r Meigen and suggested t h a t the dye m o l e c u l e may r e a c t w i t h e i t h e r the n u c l e i c a c i d or p r o t e i n m o i e t y of the gene m o l e c u l e . Evidence from the s t u d y suggested t h a t p y r o n i n (a t h i a z i n e dye) produces a g e n e t i c e f f e c t by c o m b i n i n g d i r e c t l y w i t h n u c l e i c a c i d . He a l s o suggested t h a t the mutagenic a c t i v i t y of a dye i s r e l a t e d t o i t s a f f i n i t y f o r u n p o l y m e r i z e d n u c l e i c a c i d . I n t h i s s t u d y , he r e p o r t e d t h a t n e u t r a l red was c a p a b l e of p r o d u c i n g 0.33$ l e t h a i s and t h a t rhodamine appears t o be d e f i n i t e l y a l t h o u g h w e a k l y mutagenic c a u s i n g 0.27$ l e t h a l s . The m u t a g e n i c i t y of a c r i d i n e compounds i n d i f f e r e n t b i o l o g i c a l systems was r e v i e w e d by Nasim and B r y c h c y (26). A c r i f l a v i n e and a c r i d i n e orange were r e p o r t e d t o i n c r e a s e s e x - l i n k e d and second chromosome r e c e s s i v e l e t h a l m u t a t i o n s i n both m a l e s and f e m a l e s o f D. melanagaster. I n the s i l k w o r m , Bombyx m o r l (L.), a c r i d i n e orange produced mutagenic e f f e c t s i n e g g - c o l o r l o c i i n f e m a l e but not male pupae. However, mutagenic e f f e c t s were observed w i t h p a r e n t a l chromosomes i n m i t o t i c cleavage n u c l e i . B l a n c h i e t a l . (27) s t u d i e d t h e e f f e c t s o f methylene blue on f i x e d e u k a r i o t i c chromosomes of the m o s q u i t o C u l i s e t a l o n g i a r e o l a t a under a e r o b i c c o n d i t i o n s w i t h v i s i b l e l i g h t i r r a d i a t i o n . They found t h a t d i l u t e d s o l u t i o n s of the dye d r a m a t i c a l l y a l t e r e d chromosomal s t r u c t u r e . R e s u l t s o f t h i s s t u d y suggested t h a t e l e c t r o n i c a l l y e x c i t e d Op, a s p e c i f i c p r o d u c t of the i n t e r a c t i o n among v i s i b l e l i g h t , methylene b l u e , and 0 , may be r e s p o n s i b l e f o r chromosomal DNA a l t e r a t i o n . S i m i l a r c o n c l u s i o n s were drawn by G rue ne r and Lockwood (28) i n a study on photodynamic m u t a g e n i c i t y o f r o s e bengal i n Chinese h a m s t e r embryo c e l l s . Commercial rhodamine 6G and rhodamine Β have been shown t o induce r e v e r s i o n m u t a t i o n s i n S a l m o n e l l a and s i n g l e - s t r a n d b r e a k s i n Chinese ham s t e r ovary c e l l s (29). However, a n o c h l o r 1254-induced r a t l i v e r homogenate (S9) i s r e q u i r e d f o r p r o d u c t i o n of g e n e t i c a c t i v i t y by these dyes. The photomutagenic e f f e c t s o f c h l o r promazine i n 5 Q

2

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S a l m o n e l l a and Chinese h a m s t e r ovary c e l l s were s t u d i e d by Ben-Hur e t a l . (30). They found a pH r e l a t e d e f f e c t w h i c h f a c i l i t a t e d b i n d i n g o f t h i s p h o t o t o x i n t o DNA, RNA, and p r o t e i n s w i t h i n t h e c e l l s t h a t enhanced p h o t o t o x i c i t y and m u t a g e n i c i t y . Plant-derived f u r a n o q u i n o l i n e s and c e r t a i n t r y p t o p h a n - d e r i v e d a l k a l o i d s were shown t o i n h i b i t m i t o s i s and t o cause chromosomal a b e r r a t i o n s i n m i c r o o r g a n i s m s ( b a c t e r i a , f u n g i ) and i n Chinese h a m s t e r ovary cells (υ). E f f e c t s a t the C e l l u l a r L e v e l B e s i d e s some of the s u b t l e t o the more o b v i o u s , d r a m a t i c e f f e c t s p h o t o d y n a m i c a l l y a c t i v e compounds have on c e l l u l a r components, s e v e r a l p h o t o s e n s i t i z e r s can cause c o m p l e t e d e s t r u c t i o n o f c e l l s . D e s t r u c t i o n most l i k e l y b e g i n s w i t h the a l t e r e d p e r m e a b i l i t y of the c e l l membrane w h i c h i n t u r n a l l o w s i n t r a c e l l u l a r pertubation the demise of the e n t i r t o the c y t o p l a s m w i t h subsequen lysing complet d e s t r u c t i o n ) . A d d i t i o n a l l y , the c e l l u l a r s i t e of damage and/or mode of damage i s a p p a r e n t l y dependent on the s e n s i t i z e r and i t s localization. There a r e a number of r e p o r t s on t h e e f f e c t s o f p h o t o s e n s i t i z e r s on whole c e l l s o f o r g a n i s m s o t h e r t h a n i n s e c t s (see r e v i e w a r t i c l e s ) . Blum (_1), i n h i s r e v i e w of photodynamic a c t i o n , c i t e s f i n d i n g s by v a r i o u s w o r k e r s on e f f e c t s of p h o t o t o x i n s on mammalian blood c e l l s . Under l i g h t c o n d i t i o n s , e r y t h r o c y t e s were hemolyzed and reduced i n number by s e v e r a l xanthene dyes. H e m o l y s i s a l s o was observed i n t h e absence of i r r a d i a t i o n by h i g h c o n c e n t r a t i o n s o f rose bengal. More complex changes have been n o t e d i n t o t a l l e u c o c y t e c o u n t s under i n v i t r o i r r a d i a t i o n ; numbers may g r a d u a l l y i n c r e a s e f r o m a normal l e v e l t o a c o n d i t i o n o f l e u c o c y t o s i s f o l l o w e d by l e u c o p e n i a . The r e v i e w by P o o l e r and Valenzeno (14) d i s c u s s e s photodyna mic i n a c t i v a t i o n of e r y t h r o c y t e s . Membrane p h o t o m o d i f i c a t i o n has been e x t e n s i v e l y s t u d i e d i n t h e s e c e l l s . I n t h e presence of an a p p r o p r i a t e s e n s i t i z e r and l i g h t , t h e r e i s p r o g r e s s i v e c e l l s w e l l i n g e v e n t u a l l y c u l m i n a t i n g i n l y s i s w i t h r e l e a s e of c e l l c o n t e n t s ; the s w e l l i n g and l y s i s a r e s a i d t o be of a c o l l o i d osmotic nature. No s i m i l a r o b s e r v a t i o n s have been made on i n s e c t hemocytes. G i v e n the m o r p h o l o g i c a l and presumed f u n c t i o n a l d i v e r s i t i e s o f hemocytes found f r e e i n the hemolymph and a s s o c i a t e d w i t h h e m o p o i e t i c t i s s u e o r organs, some i n t e r e s t i n g o b s e r v a t i o n s a w a i t r e s e a r c h e r s who undertake s t u d i e s o f the e f f e c t s photodyna mic a c t i o n have on these c e l l s i n i n s e c t s . I n t h e o n l y known s t u d y of the e f f e c t s o f p h o t o s e n s i t i z e r s on i n s e c t hemocytes, Weaver e t a l . (32) showed t h a t e r y t h r o s i n e Β s i g n i f i c a n t l y a f f e c t e d t h e a g g r e g a t e o f hemocytes i n the A m e r i c a n c o c k r o a c h ( P e r i p l a n e t a a m e r i c a n a (L.)). L i g h t - e x p o s e d , dyei n j e c t e d roaches showed d i m i n i s h i n g numbers o f hemocytes a t dosages f r o m 0.068 and 0.244 mg of dye/g of body w e i g h t , r e s u l t i n g i n r e d u c t i o n s o f 11 and 40$, r e s p e c t i v e l y . L i g h t - e x p o s e d , d y e - f e d r o a c h e s a l s o tended t o have f e w e r hemocytes than u n t r e a t e d controls. Roaches h e l d i n d a r k n e s s and e i t h e r d y e - f e d o r dye-

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i n j e c t e d tended t o have h i g h e r l e v e l s o f hemocytes i n a l l cases e x c e p t a t the h i g h e s t i n j e c t e d dose of 0.244 mg; a t t h i s dose, t h e r e was a s i g n i f i c a n t r e d u c t i o n o f n e a r l y 25$. The reason(s) f o r i n c r e a s e d l e v e l s i n roaches not i r r a d i a t e d a r e not known and s i m i l a r phenomena have not been noted i n mammalian systems. I t was suggested t h a t the dye a l o n e may have a f f e c t e d the a d h e s i o n o f c e l l s (e.g. c y s t o o y t e s ) t o t i s s u e , t h u s d r i v i n g n o r m a l l y none!roul a t i n g hemocytes i n t o c i r c u l a t i o n . T h i s study d i d not d e t e r m i n e i f l y s i s was t h e cause of the decreased number of hemocytes observed. The p o s s i b i l i t y was mentioned t h a t the d y e / l i g h t t r e a t ment may have caused an I n c r e a s e i n t h e number of a d h e r i n g hemoc y t e s r a t h e r than c a u s i n g l y s i s . When c o n s i d e r i n g the a g g r e g a t e o f c e l l s and t h e changes noted, the dye a p p a r e n t l y induced an i n i t i a l c o n d i t i o n o f l e u c o c y t o s i s t h a t progressed t o a s t a t e o f l e u c o p e n i a w i t h i n c r e a s e d dosage upon i r r a d i a t i o n ; s i m i l a r o b s e r v a t i o n s have been noted i n mammalian systems. One of the more commo t h a t has been observed i e v i d e n c e of l y s i n g o f midgu e p i t h e l i a fly S c h i l d m a c h e r (34) (as c i t e d by R e s p l c i o and H e i t z ( 3 5 ) ) observed c o n s i d e r a b l e d e s t r u c t i o n o f the midgut w a l l i n m o s q u i t o l a r v a e t r e a t e d w i t h a c r i d i n e r e d . C a r p e n t e r and H e i t z (36). i n t h e i r study on l a t e n t t o x i c i t y of rose bengal on l a r v a e of C u l e x p i p i e n s q u i n q u e f a s c i a t u s Say observed t h a t the gut t r a c t i n t r e a t e d l a r v a e a p p e a r e d destroyed. The c o a g u l a t i o n o f i n s e c t hemolymph appears t o be a f f e c t e d by p h o t o s e n s i t i z e r s . I n t h e i r study on hemocytes, Weaver e t a l . (32) observed t h a t g e l a t i o n o f the plasma was o f t e n a d v e r s e l y a f f e c t e d i n e r y t h r o s i n B - t r e a t e d roaches ( u n p u b l i s h e d r e s u l t s ) . Treated roaches b l e d more f r e e l y w i t h l e s s c o a g u l a t i o n t h a n d i d u n t r e a t e d c o n t r o l s . T h i s c o u l d be a d i r e c t r e s u l t of d i m i n i s h e d numbers o f c y s t o o y t e s i n hemolymph; c y s t o o y t e s have been shown t o be a s s o c i a t e d w i t h c o a g u l a b i l i t y o f the b l o o d i n P e r l p l a n e t eu E f f e c t s a t the Systems L e v e l I n f l u e n c e on Components o f Body F l u i d s . B i o c h e m i c a l changes i n I n s e c t s induced by p h o t o t o x i n s have been s t u d i e d by o n l y a few workers. Broome e t a l . (37) conducted i n v i v o s t u d i e s o f the b i o c h e m i c a l changes a s s o c i a t e d w i t h the dark r e a c t i o n o f d i e t a r y rose bengal i n t h e b o l l w e e v i l . I n c l u s i o n o f rose bengal i n t h e d i e t of newly-emerged b o l l w e e v i l s f o r f o u r days decreased l e v e l s o f t o t a l l i p i d s (90$) and t o t a l p r o t e i n s (41$) when compared t o c o n t r o l s . The t o t a l amino a c i d p o o l i n c r e a s e d 3$; l y s i n e , g l y c i n e , t y r o s i n e , h i s t i d i n e , a r g i n i n e and p r o l i n e i n c r e a s e d , whereas the r e m a i n i n g amino a c i d s e i t h e r decreased o r r e m a i n e d the same. I n a r e l a t e d paper, C a l l ah am e t a l . (38). u s i n g d i e t a r y rose bengal, s t u d i e d s i m i l a r b i o c h e m i c a l parameters i n the a d u l t b o l l w e e v i l through f i v e c o n s e c u t i v e days post-emergence. I n t h e c o n t r o l s , p r o t e i n l e v e l s n e a r l y doubled a t two days post-emergence t h e n remained f a i r l y c o n s t a n t f r o m 2-5 days; t r e a t e d a d u l t s m a i n t a i n e d the same l e v e l t h r o u g h the f i v e day p e r i o d . Amino a c i d p o o l s i z e s a t f i v e days f o r t h e c o n t r o l s and t r e a t e d a d u l t s showed d e c r e a s e s o f 14 and 20$, r e s p e c t i v e l y , when compared t o c o n t r o l s

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a t day 0. Enzyme a c t i v i t y of l a c t i c dehydrogenase and a c e t y l c h o l i n e s t e r a s e a l s o showed a decrease i n t r e a t e d w e e v i l s . I n b o t h s t u d i e s , the a u t h o r s suggest t h a t r o s e bengal can cause a l e t h a l energy s t r e s s on the organism. Hemolymph p r o t e i n s of e r y t h r o s i n B - t r e a t e d a d u l t A m e r i c a n c o c k r o a c h e s have been s t u d i e d by p o l y a c r y l a m i d e d i s c e l e c t r o p h o r e s i s (Weaver, J . E., West V i r g i n i a U n i v e r s i t y a t Morgantown, u n p u b l i s h e d data). P r o t e i n p a t t e r n s i n d y e - s e n s i t i z e d roaches were a l t e r e d i n p o s i t i o n and numbers and the mean c o n c e n t r a t i o n of p r o t e i n w i t h i n s p e c i f i c bands was s i g n i f i c a n t l y d i f f e r e n t f r o m u n t r e a t e d roaches. There were d i s t i n c t d i f f e r e n c e s between sexes and i r r a d i a t e d v e r s u s d a r k - t r e a t e d roaches. I n f l u e n c e on Body F l u i d s . The xanthene dyes, rose bengal and e r y t h r o s i n Β have been shown t o cause v o l u m e t r i c changes i n the hemolymph and c r o p c o n t e n t s o f d y e - s e n s i t i z e d A m e r i c a n and o r i e n t a l cockroaches ( B l a t t o r i e n t a i (39) Dietar i n j e c t e d dye were both e f f e c t i v i n hemolymph and c r o p volume e s p e c i a l l y i n c r o p volumes, i n i n j e c t e d roaches. I n i r r a d i a t e d roaches, hemolymph volumes p r o g r e s s i v e l y decreased and crop volumes i n c r e a s e d o v e r t i m e up t o 63 minutes. I t was suggested i n t h i s study t h a t c e l l membrane p e r m e a b i l i t y may have been a f f e c t e d thereby c r e a t i n g a d i f f e r e n t i a l i n osmotic pressure which allowed hemocoel f l u i d s t o pass i n t o the a l i m e n t a r y c a n a l . Changes i n t h e s p e c i f i c g r a v i t y of hemolymph i n the A m e r i c a n c o c k r o a c h have been observed a f t e r p h o t o s e n s i t i z a t i o n w i t h e r y t h r o s l n B. I r r a d i a t e d , s e n s i t i z e d roaches showed i n c r e a s e s i n s p e c i f i c g r a v i t y o f 0.44 and 0.81$ a f t e r 30 and 60 m i n u t e s of l i g h t response, r e s p e c t i v e l y (Amrine, J. W. J r . , West V i r g i n i a U n i v e r s i t y a t Morgantown, u n p u b l i s h e d data). These changes c o u l d be r e l a t e d t o a l o s s o f w a t e r through the M a l p i g h i a n t u b u l e s i n t o the a l i m e n t a r y c a n a l w i t h a c o n c u r r e n t i m p a i r m e n t ( f a i l u r e ) of the w a t e r r e t r i e v a l system i n the l o w e r M a l p i g h i a n t u b u l e s and r e c t a l membrane p r e c i p i t a t e d by the photodynamic a c t i o n . I n f l u e n c e on the Nervous System. S t u d i e s on a number o f o r g a n i s m s have shown t h a t e x c i t a b l e c e l l s a r e s u s c e p t a b l e t o photomodification. Normal i m p u l s e p r o p a g a t i o n and subsequent e v e n t s t r i g g e r e d by the i m p u l s e may become b l o c k e d or the i m p u l s e d i s t o r t e d i n complex ways I n s e n s i t i z e d c e l l s depending on t h e l i g h t dose. P o o l e r and Valenzeno (14) p r o v i d e a good r e v i e w o f photodynamic i n a c t i v a t i o n o f e x c i t a b l e c e l l s i n nerve axons, s k e l e t a l m u s c l e , c a r d i a c and smooth muscle i n v a r i o u s o r g a n i s m s o t h e r than i n s e c t s (see a l s o p r o c e e d i n g c h a p t e r ) . Kondo and K a s a i (40) s t u d i e d the p h o t o i n a c t i v a t i o n o f s a r c o p l a s m i c r e t i c u l u m v e s i c l e membranes o f r a b b i t by s e v e r a l xanthene dyes ( e r y t h r o s l n B, e o s i n Y, rhodamine B, methylene b l u e , r o s e bengal). They observed t h a t some r e g u l a r r e l a t i o n s h i p s e x i s t between t h e m o l e c u l a r s t r u c t u r e s of xanthene dyes and the i n a c t i v a t i o n o f these e x c i t a b l e c e l l s . Food, drug and c o s m e t i c dyes o f the xanthene t y p e have been shown under dark c o n d i t i o n s t o a c t i n a dose-dependent manner when a p p l i e d t o i s o l a t e d m o i l us can g a n g l i a ; t h e s e dyes a l t e r the p o t a s s i u m p e r m e a b i l i t y of the membrane

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t h e r e b y i n c r e a s i n g t h e r e s t i n g membrane p o t e n t i a l and conductance of the neurons (41). A l a t e r study by A u g u s t i n e and L e v i t o n (42) showed t h a t l i g h t i n t e n s i t y was a l s o a f a c t o r i n t h e degree of a c t i v i t y observed w i t h e r y t h r o s l n Β on t h e p r e s y n a p t i c e f f e c t o f t h i s xanthene dye a t the f r o g n e u r o m u s c u l a r j u n c t i o n . I n a study on the s y n a p t i c c o n n e c t i v i t y between a u d i t o r y i n t e r n e u r o n s of the c r i c k e t , G r y l l u s b l m a c u l a t u s DeGeer, S e l v e r s t o n e t a l . (43) used the i n t r a c e l l u l a r dye, l u c i f e r y e l l o w , t o s e l e c t i v e l y p h o t o i n a c t i v a t e s i n g l e neurons i n p r o t h o r a c i c g a n g l i o n . Various b e h a v i o r i a l responses observed i n i n s e c t s subjected t o p h o t o r e a c t i v e s u b s t a n c e s s t r o n g l y suggest t h a t xanthene dyes, e s p e c i a l l y , can s e r i o u s l y a f f e c t the nervous system. Yoho e t a l . (44) o b s e r v e d t h a t s e n s i t i z e d house f l i e s a f t e r a dark p e r i o d were i n i t i a l l y v e r y a c t i v e when f i r s t exposed t o l i g h t . Periods of h y p e r a c t i v i t y , c h a r a c t e r i z e d by s p o r a d i c b u r s t s o f f l y i n g and p r o l o n g e d a n t e n n a l and w i n g - c l e a n i n g movements, were f o l l o w e d by p e r i o d s o f quiescence. Prolonge associated with regurgitatio movements became u n c o o r d i n a t e l e g s ; s i m u l t a n e o u s l y , o v i p o s i t o r s of f e m a l e s were o f t e n o s b e r v e d i n an extended p o s i t i o n . F l i e s became p r o g r e s s i v e l y u n c o o r d i n a t e d , o f t e n f a l l i n g o n t o t h e i r s i d e or dorsum. Many f l i e s d i e d w i t h l e g s f o l d e d v e n t r a i l y o v e r the t h o r a x ; o t h e r s d i e d i n a n o r m a l u p r i g h t p o s i t i o n . Broome e t a l . (45) noted t h a t i m p o r t e d f i r e a n t s s e n s i t i z e d w i t h rose bengal e x h i b i t e d i n c r e a s e d i r r i t a b i l i t y , i n c r e a s e d a n t e n n a l grooming, l o s s o f l o c o m o t o r y c o o r d i n a t i o n , f o l l o w e d by a t e t a n i c p a r a l y s i s p r i o r t o death. The a n t s q u i t e o f t e n assumed "a c o n t o r t e d p o s i t i o n a t death c h a r a c t e r i z e d by p o s i t i o n i n g t h e abdomen under the t h o r a x w i t h t h e c e p h a l i c r e g i o n drawn down." Other E f f e c t s D e v e l o p m e n t a l Aspects. I n a r e v i e w by Barbosa and P e t e r s (46), s e v e r a l r e p o r t s ( p r i o r t o 1971) a r e n o t e d w h i c h i n c l u d e d o b s e r v a t i o n s on adverse e f f e c t s o f p h o t o a c t i v e s u b s t a n c e s on i n s e c t development. G r o w t h - r a t e r e t a r d a t i o n appears t o be one o f t h e more commonly observed e f f e c t s . P r o l o n g e d p e r i o d s o f l a r v a l development and u n d e r s i z e d pupae have been r e p o r t e d . Some w o r k e r s s p e c u l a t e d i n t h e s e e a r l y s t u d i e s , and more r e c e n t l y (37-38), t h a t p h o t o t o x i n s i n h i b i t maximum u t i l i z a t i o n of energy s o u r c e s or cause l e t h a l energy s t r e s s e s i n t h e organism. I n more r e c e n t s t u d i e s , decreased body w e i g h t s of I n s e c t s s e n s i t i z e d w i t h s u b s t i t u t e d xanthene dyes have been documented. C o r r e l a t i o n s between the decrease i n body w e i g h t i n the b o l l w e e v i l and e f f i c i e n c y of dyes ( i n c r e a s i n g h a l o g e n a t i o n ) were r e p o r t e d by C a l l a h a m e t a l . (47) as rose b e n g a l > p h l o x i n Β > e r y t h r o s l n Β > e o s i n Y. F u r t h e r d o c u m e n t a t i o n o f rose bengal a f f e c t i n g body w e i g h t i n the b o l l w e e v i l was made by Callaham e t a l . (38); u n t r e a t e d i n s e c t s showed w e i g h t g a i n s o f 30$ w h i l e w e i g h t s of t r e a t e d w e e v i l s r e m a i n e d e s s e n t i a l l y the same. G r o w t h i n h i b i t i o n i n t h e house f l y t r e a t e d w i t h rose bengal and e r y t h r o s l n Β has been i n v e s t i g a t e d (48); l a r v a e r e a r e d on t r e a t e d medium showed i n h i b i t i o n o f p u p a t i o n and decreased p u p a l w e i g h t s .

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129

Clement e t a l . (49) examined t h e e f f e c t o f rose b e n g a l on development o f l a r v a e o f t h e b l a c k cutworm ( A g r o t i s I p s i l o n (Hufnagel)). U s i n g t h e number o f f e c a l p e l l e t s produced, they found t h a t t r e a t e d l a r v a e produced s i g n i f i c a n t l y f e w e r p e l l e t s t h a n c o n t r o l s . Retarded l a r v a l g r o w t h observed i n t h i s study may have been t h e r e s u l t o f t e m p o r a r y i n h i b i t i o n o f i n g e s t i o n . I n s e c t g r o w t h r e g u l a t o r s (IGRs) a r e c u r r e n t l y a new t e c h n o l o g y b e i n g developed f o r i n s e c t c o n t r o l . There i s c o n s i d e r a b l e evidence t h a t some p h o t o t o x i n s a f f e c t i n s e c t s s i m i l a r l y . Barbosa and P e t e r s (46) i n t h e i r r e v i e w m e n t i o n d e s c r i p t i o n s o f developmental a b n o r m a l i t i e s of s e n s i t i z e d i n s e c t s r e s e m b l i n g those t h a t more r e c e n t l y have been n o t e d i n I G R - t r e a t e d i n s e c t s . N e u t r a l red was r e p o r t e d t o cause 80$ m o r t a l i t y i n e l a t e r i d l a r v a e a s t h e m o u l t i n g phase began (50). Morphological a b b e r r a t i o n s were noted i n l a r v a e o f t h e b u t t e r f l y C o l l a s eurytheme (Bols.) when f e d d i e t s o f n e u t r a l r e d . In a related s p e c i e s (Ç. p h i l o d i c e (L.)) deformations. Bridges e pound a g a i n s t l a r v a e o aegypt produce morphogenetic e f f e c t s s i m i l a r t o methoprene. L a r v a e f a i l e d t o c o m p l e t e t h e m o u l t i n g p r o c e s s and l a r v a l - p u p a l i n t e r m e d i a t e s were formed. Specimens t h a t formed a p p a r e n t l y normal pupae o f t e n d i e d b e f o r e t h e a d u l t emerged o r d i e d o n l y p a r t i a l l y emerged. A d u l t males t h a t emerged n o r m a l l y d i d n o t complete g e n i t a l i a r o t a t i o n . M o r p h o l o g i c a l a b n o r m a l i t i e s i n l a r v a e and pupae f r o m rose b e n g a l t r e a t e d m o s q u i t o l a r v a e have been s u g g e s t e d t o r e s u l t f r o m i m p r o p e r f o r m a t i o n o f c h i t i n (52) o r f r o m a dye-induced energy s t r e s s on t h e i n s e c t (36). The IGR e f f e c t has been observed i n pupae o f t h e f a c e f l y when l a r v a e were t r e a t e d w i t h rose bengal and e r y t h r o s l n Β (53). Most f l i e s d i e d i n t h e p u p a l s t a g e as t h e a d u l t a t t e m p t e d t o emerge f r o m t h e puparium; some m o r p h o l o g i c a l l y normal f l i e s emerging f r o m e r y t h r o s l n B - t r e a t e d manure had s h o r t e r l i f e spans t h a n t h e c o n t r o l s . Downum e t a l . (54), i n a study on t h e p h o t o t o x i c e f f e c t s o f a l p h a - t e r t h i e n y l on t h e t o b a c c o hornworm (Manduca s e x t a (L.)), noted d r a s t i c d e v e l o p m e n t a l a l t e r a t i o n s i n t r e a t e d l a r v a e . Dietary alpha-terthienyl, with i r r a d i a t i o n of larvae a f t e r i n g e s t i o n , r e s u l t e d i n d e l a y e d and abnormal pupal f o r m a t i o n w i t h no subsequent a d u l t emergence; a d d i t i o n a l l y , l a r v a l g r o w t h was d e l a y e d f o r f o u r days a f t e r t r e a t m e n t when l a r v a e r e f u s e d d i e t . Pronounced t i s s u e n e c r o s i s was observed a t a p p l i c a t i o n s i t e s o f t o p i c a l l y t r e a t e d , i r r a d i a t e d l a r v a e ; a t p u p a t i o n , normal s c l e r o t i z a t i o n and i n d e n i z a t i o n were a f f e c t e d i n v a r i o u s a r e a s o f t h e p u p a l case. R e p r o d u c t i v e Aspects. There a r e a p p a r e n t l y o n l y a few r e p o r t s of the adverse e f f e c t s o f photodynamically a c t i v e s u b s t a n c e s on t h e r e p r o d u c t i v e p o t e n t i a l of i n s e c t s . I n an e a r l y r e p o r t , David (55) s p e c u l a t e d t h a t methylene b l u e was "somehow a f f e c t i n g gametogenesis" o f D r o s o p h l l a i n a study o f t h e e f f e c t s o f t h i s dye on s u c c e s s i v e g e n e r a t i o n s . I n a l a t e r study, David (56) observed t h a t methylene b l u e caused a marked decrease by a f a c t o r o f f o u r i n the f e c u n d i t y o f t r e a t e d Drosophlla females. More r e c e n t l y , P i m p r i k a r e t a l . (57) r e p o r t e d t h a t f e c u n d i t y

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i n t h e house f l y was d i r e c t l y r e l a t e d t o t h e d i e t a r y c o n c e n t r a t i o n o f rose bengal and t h e frequency of dye f e e d i n g ; up t o 10% reduc t i o n i n f e c u n d i t y was observed i n f e m a l e s m a i n t a i n e d on a c o n t i n u o u s d i e t f o r 15 days. As noted i n t h e p r e v i o u s s e c t i o n , s e v e r a l s t u d i e s have shown t h a t pupal and a d u l t w e i g h t s o f some i n s e c t s are decreased by t r e a t m e n t s w i t h xanthene dyes. However, no o b s e r v a t i o n s were made i n these s t u d i e s on how these developmental a b n o r m a l i t i e s might a f f e c t reproduction. Since i t i s known f o r some I n s e c t s p e c i e s t h a t a c o r r e l a t i o n e x i s t s between p u p a l w e i g h t and number of eggs produced by f e m a l e s , i t seems r e a s o n a b l e t o assume t h a t s e n s i t i z e d i n s e c t s p r o d u c i n g a b n o r m a l l y s m a l l e r pupae o r a d u l t s w h i c h f a i l t o show normal w e i g h t g a i n s may not be capable of p r o d u c i n g a normal complement of eggs. O v i c i d a l p r o p e r t i e s of p h o t o t o x i n s have not been s t u d i e d e x t e n s i v e l y . P i m p r i k a r e t a l . (57) i n v e s t i g a t e d the o v i c i d a l a c t i v i t y of s i x xanthene d e r i v a t i v e s a g a i n s t the house f l y . Rose bengal and e r y t h r o s l n Β a c t i v causin 30$ inhi b i t i o n of egg h a t c h w h i l a c t i v e c a u s i n g about 15 observed by the a u t h o r s were: some eggs f a i l e d t o h a t c h (presumably due t o embryo death p r i o r t o hatch); some l a r v a e e c l o s e d f r o m a l o n g i t u d i n a l l i n e o f weakness a t the me s a l dorsum of the c h o r i o n ; some l a r v a e f r e e d t h e head c a p s u l e , but were u n a b l e t o f r e e the c a u d a l end from the c h o r i o n . Kagan and Chan (58) s t u d i e d t h e p h o t o o v i c i d a l a c t i v i t y of t h r e e n a t u r a l l y o c c u r r i n g m o l e c u l e s a g a i n s t D. melanogaster. P h e n y l h e p t a t r i y n e , a l p h a - t e r t h i e n y l , and 8-methoxypsoralan a l l prevented egg h a t c h . The f i r s t two were t o x i c t o eggs i n the dark, but upon i r r a d i a t i o n , e f f e c t i v e n e s s was I n c r e a s e d 37 and 4,333-fold, r e s p e c t i v e l y . Summary and Remarks I n g e n e r a l , we know f a r l e s s about t h e p h y s i o l o g i c a l e f f e c t s o f photodynamic a c t i o n on i n s e c t s t h a n we do about t h i s phenomenon as i t a f f e c t s t h e p h y s i o l o g y o f o t h e r i n v e r t e b r a t e s and mammals. T h i s i s e s p e c i a l l y t r u e a t the s u b c e l l u l a r and c e l l u l a r l e v e l s . At t h e systems l e v e l , we have gained c o n s i d e r a b l e knowledge d u r i n g the l a s t decade of how p h o t o t o x i n s can m o d i f y the b i o c h e m i c a l processes and p h y s i o l o g y of i n s e c t s . But s t i l l , o n l y a l i m i t e d number of s t u d i e s have been done a t t h i s l e v e l , p a r t i c u l a r l y w i t h t h e nervous system; a t t h i s p o i n t i n t i m e we can o n l y s p e c u l a t e from abnormal b e h a v i o r a l p a t t e r n s observed i n s e n s i t i z e d i n s e c t s t h a t p e r t u b a t i o n s a r e o c c u r r i n g i n t h e nervous system. There i s s t r o n g e v i d e n c e f r o m r e c e n t s t u d i e s t h a t some pho t o a c t i v e s u b s t a n c e s can m o d i f y the p h y s i o l o g i c a l processes i n i n s e c t s i n much the same manner as IGRs. There appears t o be a need f o r f u r t h e r study on how the p h o t o t o x i n s a f f e c t t h e r e p r o d u c t i v e p o t e n t i a l of i n s e c t s ; many IGRs have been shown t o reduce f e c u n d i t y and induce s t e r i l i t y , but o n l y a c o u p l e o f r e c e n t s t u d i e s have d e a l t w i t h t h i s a s p e c t i n any d e t a i l u s i n g the more e f f e c t i v e p h o t o t o x i n s . I n s t u d i e s where pupal and a d u l t w e i g h t s

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were observed t o be a l t e r e d I n s e n s i t i z e d i n s e c t s , t h e r e was no f o l l o w u p a s t o what e f f e c t these a b n o r m a l i t i e s may have on r e p r o ductive ability. I t has been suggested t h a t the photodynamic a c t i o n mechanism may not be a v i a b l e o p t i o n f o r i n s e c t c o n t r o l (15). Recognizing t h a t a l l the easy c h e m i s t r y on i n s e c t i c i d e s has been done, i s n ' t i t time t o explore phototoxins as an a l t e r n a t i v e t o , o r p o s s i b l e use i n a n a d j u n c t i v e r o l e , t o the more c o n v e n t i o n a l i n s e c t i c i d e s ? Whatever some may t h i n k about i n v e s t i g a t i n g " i n s i d i o u s and l i t t l e understood mechanisms (of p h o t o t o x i n s ) t o rescue ... a b o r d e r l i n e t e c h n o l o g y " (15). i t s h o u l d r e m a i n a c h a l l e n g e t o r e s e a r c h e r s t o d e v e l o p t h a t t e c h n o l o g y t o combat I n s e c t p e s t s . The more we understand about the modes o f a c t i o n o f p h o t o d y n a m i c a l l y a c t i v e s u b s t a n c e s , t h e more i n t e l l i g e n t l y we w i l l be a b l e t o use them t o our b e n e f i t .

Literature Cited 1.

2. 3. 4. 5. 6. 7.

8. 9. 10. 11.

12. 13. 14. 15.

Blum, H. F. Photodynami y Light; Reinhold Publ. Corp.: New York, 1941; 309 p. (Reprinted in 1964 with an updated appendix by Hafner Publ., New York). Errera, M. Progr. Biophys. Biophys. Chem. 1953, 3, 88-130. Clare, N. t. In Radiation Biology; Holleander, Α., Ed.; McGraw-Hill: New York, 1956; Vol. III, pp 693-723. Fowlk, W. L. J . Invest. Dermatol. 1959, 32, 233. Santamaria, L. In Recent Contributions to Cancer Research in Italy. Tumari Suppl.; Bucalossi, P.; Veroneri, U., Eds.; Casa Editrice Ambrosiana: Milan, 1960; pp 167-287. Santamaria, L. Bull. Sol. Chim. Belges. 1962, 71, 889. Santamaria, L.; Pinto, G. In Research Progress in Organic. Biological and Medicinal Chemistry; Gallo, U; Santamaria, L., Eds.; Soc. Editorial a Farmaceutica: Milan, 1964; Vol. 3, pp 259-336. Spikes, J. D.; Ghiron, C. A. In Physical Processes in Radiation Biology; Augenstein, L. G.; Mason, R; Rosenberg, B., Eds.; Academic Press: New York, 1964; pp 309-336. Simon, M. I. In Comprehensive Biochemistry; Florkin, M.; Stotz, Ε. H., Eds.; Elesevier: Amsterdam, 1967, Vol. 27, pp 137-56. Spikes, J . D.; Straight, R. Ann. Rev. Phys. Chem. 1967, 18, 409-36. Bourdon, J . ; Schnurizer, B. In Physics and Chemistry of the Organic Solid State; Fax. D.; Labes, M. M.; Weissberger, Α., Eds.; Wiley (Interscience): New York, 1967, Vol. 3, PP 59-131. Spikes, J. D. In Photophysiology III. Current Topics; Giese, A. C., Ed.; Academic Press: New York, 1968; pp 36-64. Spikes, J . D.; Livingston, R. Adv. Rad. Biol. 1969, 3, 29-121. Pooler, J . P.; Valenzeno, D. P. Med. Phys. 1981, 8, 614-28. Robinson, J . R. Res. Rev. 1983, 88, 69-100.

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18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44.

LIGHT-ACTIVATED PESTICIDES Cande, W. Z.; McDonald, K.; Meeusen, R. L. J . C e l l B i o l . 1981, 88. 618-29. Haga, J . Y.; Spikes, J . D. Research Progress in Organic, Biological and Medicinal Chemistry; Galo, U; Santamaria, L., Eds.; American Elsevier Publishing Co.: New York, 1972; Vol. 3, pp 464-79. H i l f , R.; Small, B. D.; Murant, S. R.; Leakey, B. P.; Gibson, L. S. Cancer Res. 1984, 44, 1483-88. Palm, Ν. B. Ark. Zool. Stockholm. Series II 1952, 3, 195-272. Carpenter, T. L.; Mundie, T. G.; Ross, J . H.; Heitz, J . R. Environ. Entomol. 1981, 10, 953-55. Fondren, J . E. Jr.; Heitz, J . R. Ibid. 1978, 7, 843-46. Fondren, J . E. J r . ; Norment, B. R.; Heitz, J . R. Ibid. 1978, 7, 205-8. Callaham, M. F.; Lewis, L. Α.; Holloman, M. E.; Broome, J. R.; Heitz, J. R. Comp. Biochem. Physio. 1975, 51C. 123-28. Callaham, M. F.; J. R. Pestic. Biochem. Physiol. 1977, 7, 21-7. Clark, A. M. Am. Nat. 1953, 87, 295-305. Nasim, Α.; Brychcy, T. Mutation Res. 1979, 65, 261-88. Bianchi, U.; Mezzanotte, R.; Ferrucci, L . ; Marshi, A. Cell Differentiation. 1980, 9, 323-28. Gruener, N.; Lockwood, M. P. Biochem. Biophys. Res. Commun. 1979, 90, 460-65. Nestmann, E. R.; Douglas, G. R.; Matula, T. I.; Grant, C. E.; Kowbel, D. J . Cancer Res. 1979, 39, 4412-17. Ben-Hur, E.; Prager, A. Green, M. Rosenthal, I. Chem.-Biol. Interact. 1980, 29, 223-33. Towers, G. H.; Abramowski, Z. J . Nat. Prod. 1983, 46, 572-77. Weaver, J. E.; Butler, L; Amrine, J . W. Jr. Environ. Entomol. 1982, 11, 463-66. Yoho, T. P. Ph.D. Thesis, West Virginia University, West Virginia, 1972. Schildmacher, H. Biol. Zentr. 1950, 69, 468. Respicio, N.C.;Heitz, J . R. B u l l . Environ. Contam. Toxicol. 1981, 27, 274-81. Carpenter, T. T.; Heitz, J . R. Environ. Entomol. 1980, 9, 533-37. Broome, J . R.; Callaham, M. F.; Poe, W. E.; Heitz, J . R. Chem.-Biol. Interactions 1976, 14, 203-6. Callaham, M. F.; Broome, J . R.; Poe, W. E.; Heitz, J . R. Environ. Entomol. 1977, 6, 669-73. Weaver, J . E.; Butler, L . ; Yoho, T. P. Ibid. 1976, 840-44. Kondo, M.; Kasai, M. Photochem. Photobiol. 1974, 19, 35-41. Levitan, H. Proc. Natl. Acad. Sci. 1977, 74, 2914-18. Augustine, J . G.; Levitan, H. J . Physiol. 1983, 334, 65-77. Selverston, A. I.; Kleindienst, H. U.; Huber, F. J . Neurosci. 1985, 5, 1283-92. Yoho, T. P.; Weaver, J . E.; Butler, L. Environ. Entomol. 1973, 2, 1092-96.

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8. w Ε AVER 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58.

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Broome, J . R.; Callaham, M. F.; Lewis, L. Α.; Ladner, C. M.; Heitz, J. R. Comp. Biochem Physiol. 1975, 51C, 117,21. Barbosa, P.; Peters, T. M. Histochemical J . 1971, 3, 71-93. Callaham, M. F.; Broome, J. R.; Lindig, O. H.; Heitz, J . R. Environ. Entomol. 1975, 4, 837-41. Sakurai, H.; Heitz, J . R. Ibid. 1982, 11, 467-70. Clement, S. L.; Schmidt, R. S.; Szatmari-Doogman, F.; Levine, E. J . Econ. Entomol. 1980, 73, 390-92. Zacharuk, R. Y. Can. J. Zool. 1963, 41, 991-96. Bridges, A. C.; Cocke, J.; Olson, J . K.; Mayer, R. J. Mosq. News. 1977, 37, 227-31. Pimprikar, G. D.; Norment, B. R.; Heitz, J . R. Environ. Entomol. 1979, 8, 856-59. Fairbrother, T. E.; Essig, H. W.; Combs, R. L.; Heitz, J . R. Ibid. 1981, 10, 506-10. Downum, K. R., Rosenthal, G. Α., Towers, G. Η. N. Pestic. Biochem. Physiol. 1984 David, J . C . r . Acad David, J . Bull. Biol. France Belgique. 1963, 97, 515-30. Pimprikar, G. D.; Noe, B. L.; Norment, B. R.; Heitz, J . R. Environ. Entomol. 1980, 9, 785-88. Kagan, J.; Chan, G. Experientia. 1983, 39, 402-3.

RECEIVED November 20, 1986

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 9

Multiple Mechanisms of Dye-Induced Toxicity in Insects G. D. Pimprikar and Mary Jane Coign Department of Biochemistry, Mississippi State University, Mississippi State, MS 39762

Several xanthen dye prove to various species of insects. Three types of toxic mechanism have been observed in insects namely: a light dependent mechanism, a light independent mechanism and a developmental toxicity mechanism. The light dependent mechanism is quite fast and involves production of singlet oxygen. The dark reaction is comparatively slow. Both reactions cause histological, behavioral, physiological, and biochemical changes in insects. Several morphological abnormalities are caused by the dye treatment in various insect species. Xanthene dye treatment also affects growth and development in insects. Attempts have been made to review the biochemical, physiological, and developmental aspects of these multiple toxicity mechanisms.

S e v e r a l s y n t h e t i c dyes and n a t u r a l p r o d u c t s a r e known t o be t o x i c t o v a r i o u s a g r i c u l t u r a l and p u b l i c h e a l t h i n s e c t p e s t s . A f t e r e x t e n s i v e f i e l d t e s t i n g , one o f the s y n t h e t i c dyes, e r y t h r o s i n B, has been r e g i s t e r e d by the H i l t o n - D a v i s Chemical Company f o r house f l y c o n t r o l i n caged l a y e r c h i c k e n houses under the name Intercept or Synerid. I n e a r l i e r days, the t o x i c i t y was thought t o be due t o the p r o d u c t i o n o f s i n g l e t oxygen and t h a t l i g h t was an e s s e n t i a l e l e ment f o r the t o x i c i t y . However, work done by v a r i o u s r e s e a r c h e r s over the l a s t decade has shown t h a t t h e r e a r e three types o f t o x i c i t y mechanisms a s s o c i a t e d w i t h these compounds: 1. 2. 3.

L i g h t dependent t o x i c i t y mechanism L i g h t independent t o x i c i t y mechanism Developmental t o x i c i t y mechanism

0097-6156/87/0339-0134$06.00/0 © 1987 American Chemical Society

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The l i g h t dependent t o x i c i t y mechanism i s q u i t e f a s t and needs c o m p a r a t i v e l y lower c o n c e n t r a t i o n s o f the p h o t o s e n s i t i z e r and a source o f l i g h t . The l i g h t independent o r dark mechanism i s s l o w , needs a h i g h e r c o n c e n t r a t i o n o f s e n s i t i z e r and o p e r a t e s i n the absence o f l i g h t . I n the developmental t o x i c i t y mechanism, t h e i n s e c t i s exposed t o a s u b l e t h a l dose o f the compound i n the e a r l i e r stages o f development. T h i s r e s u l t s i n m o r t a l i t y o r some adverse m o r p h o l o g i c a l a b n o r m a l i t i e s d u r i n g development, such as d e l a y e d development, growth r e t a r d a t i o n , and f e c u n d i t y and f e r t i l i t y changes. L i g h t Dependent T o x i c i t y Mechanisms The l i g h t dependent t o x i c i t y mechanism ( o r photodynamic a c t i o n ) i n i n s e c t s i n v o l v e s the i n g e s t i o n o f the p h o t o s e n s i t i z e r by the i n s e c t , f o l l o w e d by exposure t o a v i s i b l e l i g h t source which r e s u l t s i n the death o compounds have been r e p o r t e i n b i o l o g i c a l systems i n c l u d i n g , , phenothiazi nes, p s o r a l e n s , f l a v i n s , p o r p h r i n s , q u i n o n e s , p o l y i n e s and thiophenes. Photodynamic a c t i o n i n v o l v e s p h o t o o x i d a t i o n o f v a r i o u s s u b s t r a tes which r e s u l t s i n i n a c t i v a t i o n o f b i o l o g i c a l systems, d i s t o r t i o n of membranes, i n a c t i v a t i o n o f enzymes, c e l l death and o t h e r s p e c i a l f u n c t i o n l o s s e s ( 1 - 4 ) . Photodynamic a c t i o n o c c u r s v i a e i t h e r a "Type I " mechanism which i n v o l v e s e l e c t r o n t r a n s f e r r e a c t i o n s o r a "Type I I " mechanism which i n v o l v e s s i n g l e t oxygen (_5) · I n heterogenous b i o l o g i c a l systems the photodynamic r e a c t i o n may not be s t r i c t l y Type I o r Type I I mechanism but i t c o u l d i n v o l v e both mechanisms. Foote has r e v i e w e d t h e Type I and Type I I mechanisms, the f a c t o r s d e t e r m i n i n g the e f f i c i e n c y , and the r e l a t i v e p a r t i c i p a t i o n o f these mechanisms i n an e a r l i e r c h a p t e r o f t h i s book. The photodynamic damage in v i v o may occur wherever an e f f i c i e n t p h o t o s e n s i t i z e r can be i n t i m a t e l y d e p o s i t e d i n an a c t i v e l y r e s p i r i n g medium and can r e c e i v e adequate i l l u m i n a t i o n (6). In i n s e c t s , photodynamic damage most p r o b a b l y o c c u r s i n the membranes o f the gut w a l l f o l l o w e d r a p i d l y by i m p l i c a t i o n o f o t h e r l i p o i d a l membranes as the h i g h l y l i p i d - s o l u b l e p h o t o s e n s i t i z e r d i f fuses throughout the organism. The p e r m e a b i l i t y o f the photosens i t i z e r i n t o the c e l l , d i s t r i b u t i o n o f t h e p h o t o s e n s i t i z e r among v a r i o u s c e l l components, and b i n d i n g o f the p h o t o s e n s i t i z e r t o t h e s u b s t r a t e determines the n a t u r e and e x t e n t o f the photodamage (]_). A c t u a l membrane p e n e t r a t i o n by t h e p h o t o s e n s i t i z e r i t s e l f may not be r e q u i r e d t o produce h i g h l e t h a l i t y when the c o n t a c t i s s u f f i c i e n t l y i n t i m a t e and i n v o l v e s a l a r g e s p e c i f i c s u r f a c e ( S) · The s i n g l e t oxygen produced i n photodynamic a c t i o n can f r e e l y d i f f u s e m i c e l l e r as w e l l as aquous phases and can r e a c t

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w i t h o r g a n i c s u b s t r a t e s at d i f f e r e n t s i t e s ( 9 - 1 0 ) . C o n s i d e r i n g the r e a c t i v i t y o f the s i n g l e t oxygen w i t h u n s a t u r a t e d l i p i d s , i t i n d i c a tes t h a t transmembrane d i f f u s i o n c o u l d s c a r c e l y take p l a c e i n the absence o f some u n d e f i n e d t r a n s p o r t mechanism w i t h o u t p e r t u b a t i o n o f membrane t r a n s p o r t ( 8 ) . Nieumint et a l (II) r e c e n t l y presented evidence t h a t the i n t e r a c t i o n of p h o t o s e n s i t i z e r and s u b s t r a t e does i n f l u e n c e a c t u a l product f o r m a t i o n . D i r e c t i n t e r a c t i o n c o u l d take p l a c e between the s e n s i t i z e r and a d j a c e n t r e s i d u e s w h i l e more d i s t a n t domains c o u l d be o x i d i z e d by d i f f u s a b l e i n t e r m e d i a t e s such as s i n g l e t oxygen ( 1 2 ) . Photodynamic a c t i o n i s known to cause n u c l e a r , r i b o s o m a l , c y t o p l a s m i c , and c e l l membrane damaging r e a c t i o n s l e a d i n g u l t i mately to c e l l death. The t h r e e primary t a r g e t s i t e s of the photodynamic a c t i o n a r e : 1. 2. 3.

1.

B i o c h e m i c a l component B i o l o g i c a l membrane V i t a l enzyme system

B i o c h e m i c a l Components

The e f f e c t of photodynamic a c t i o n on v a r i o u s b i o c h e m i c a l com ponents has been reviewed by Spikes i n an e a r l i e r c h a p t e r of t h i s book. In o r d e r t o a v o i d d u p l i c a t i o n , t h i s a r e a i s v e r y b r i e f l y summarized h e r e . The b i o c h e m i c a l f u n c t i o n a l groups which are a t t a c k e d by photodynamic a c t i o n i n c l u d e p r o t e i n s , c a r b o h y d r a t e s , s t e r o i d s , amino a c i d s ( c y s t e i n e , t r y p t o p h a n , h i s t i d i n e , t y r o s i n e , and m e t h i o n i n e ) , f a t t y a c i d s , n u c l e i c a c i d s , t h i o l s , s u l f i d e s , and d i s u l f i d e s (13). B i n d i n g o f the dye t o b i o l o g i c a l macromolecules i s c r u c i a l and may a f f e c t the r e l a t i v e e f f i c i e n c y of Type I and Type I I pathways f o r p h o t o o x i d a t i o n a v a i l a b l e t o the s e n s i t i z e r (1Λ). Secondly, pho todynamic e f f e c t s in v i v o are l a r g e l y dependent on the s i t e t o which the p h o t o s e n s i t i z e r b i n d s . Rose bengal b i n d s a t hydrophobic s i t e s and l y s e s membranes w h i l e a c r i d i n e orange p e n e t r a t e s t o the n u c l e u s and causes damage t o DNA (7,15-16).The furanocoumarins a l s o b i n d and c r e a t e photochemical damage at the l e v e l of DNA ( 1 7 ) . The p h o t o s e n s i t i z e d o x i d a t i o n of p r o t e i n s , as w e l l as o t h e r b i o c h e m i c a l components, a l t e r s or d e s t r o y s normal b i o l o g i c a l f u n c t i o n s . I n the case of p r o t e i n s , p h o t o a l t e r a t i o n i s due t o the d e g r a d a t i o n of the s i d e c h a i n s of f i v e amino a c i d s . I n a c t i v a t i o n r e s u l t s from the d e s t r u c t i o n of e s s e n t i a l amino a c i d r e s i d u e s a t or near the a c t i v e s i t e or b i n d i n g s i t e of the enzyme and/or by the d e g r a d a t i o n of r e s i d u e s elsewhere t h a t are r e q u i r e d f o r the

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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m a i n t a i n a n c e o f the a p p r o p r i a t e c a t a l y t i c c o n f o r m a t i o n o f the m o l e c u l e (13). The i l l u m i n a t i o n o f p h o t o s e n s i t i z e r - p r o t e i n m i x t u r e s can r e s u l t i n the f o r m a t i o n o f c o v a l e n t s e n s i t i z e r - p r o t e i n photoadd u c t s which can a l t e r t h e p r o p e r t i e s o f p r o t e i n s . Photodynamic a c t i o n on n u c l e i c a c i d s r e s u l t s i n s e l e c t i v e d e s t r u c t i o n o f guanine r e s i d u e s (1_8) and a l t e r a t i o n o f the p h y s i c a l p r o p e r t i e s o f DNA. Photodynamic a c t i o n s e n s i t i z e d by rose b e n g a l can cause s t r a n d breaks i n DNA. The s e n s i t i z e r and oxygen m o l e c u l e s i n t e r a c t w i t h the DNA so as t o e f f e c t s i t e - s p e c i f i c s i n g l e t oxygen g e n e r a t i o n which causes photodynamic l e s i o n s (11) · The photosen s i t i z e d o x i d a t i o n o f r e s i d u e s i n template DNA and RNA d e c r e a s e s the e f f i c i e n c y o f t r a n s c r i p t i o n and t r a n s l a t i o n r e s p e c t i v e l y ( 4 ) . N a t u r a l l y o c c u r i n g p h o t o s e n s i t i z e r s , such as K h e l l i n and the f u r a n o coumarins, form m o n o f u n c t i o n a l adducts r e s u l t i n g i n i n t e r s t r a n d c r o s s - l i n k a g e o f DNA i n v a r i o u s developmental stages o f i n s e c t s (17,19-21). These photodynami f o r DNA t r a n s c r i p t i o n s Photodynamic a c t i o n a f f e c t s b i o l o g i c a l l y important l i p i d s i n the form o f u n s a t u r a t e d l i p i d s , such as f a t t y a c i d s , t r i g l y c e r i d e s and p h o s p h o l i p i d s ; and u n s a t u r a t e d l i p i d - s o l u b l e b i o m o l e c u l e s , such as c h o l e s t e r o l , v i t a m i n D, s t e r o l s , s t e r o i d s , and p r o s t a g l a n d i n s (Γ3). L i p i d p e r o x i d a t i o n i s q u i t e d e s t r u c t i v e t o b i o l o g i c a l membra nes. This t o p i c i s d i s c u s s e d i n d e t a i l i n the f o l l o w i n g s e c t i o n on the e f f e c t o f photodynamic a c t i o n on b i o l o g i c a l membranes. Recent s t u d i e s showed a d e p l e t i o n o f g l u t a t h i o n l e v e l s due t o the photodynamic a c t i o n i n i n s e c t s ( 2 2 - 2 3 ) . Wages (22) a l s o r e c o r d e d a d e p l e t i o n i n NADPH l e v e l s accompanied by a moderate i n c r e a s e i n NADP l e v e l s i n the p h o t o d y n a m i c a l l y t r e a t e d house f l i e s . The a u t h o r suggested t h a t , assuming some r e l a t i o n between the d e p l e t i o n o f NADPH and g l u t a t h i o n e , a t l e a s t some o f the g l u t a t h i o n e i s b e i n g o x i d i z e d t o g l u t a t h i o n e d i s u l f i d e , s i n c e the major enzyme i n v o l v e d i n m a i n t a i n i n g the e q u i l i b r i u m between g l u t a t h i o n e and g l u t a t h i o n e d i s u l f i d e , g l u t a t h i o n e r e d u c t a s e , u t i l i z e s NADPH as a donor of e l e c t r o n s f o r t h e r e d u c t i o n o f g l u t a t h i o n e . Photodynamic a c t i o n may r e s u l t i n d e p l e t i o n o f i m p o r t a n t b i o c h e m i c a l groups which a r e i n d i s p e n s a b l e t o the i n s e c t from the v i e w p o i n t o f energy m e t a b o l i s m or d e t o x i f i c a t i o n mechanisms. 2.

B i o l o g i c a l Membranes

Valenzeno and P o o l e r have reviewed the e f f e c t s o f photodynamic a c t i o n on b i o l o g i c a l membranes i n e a r l i e r c h a p t e r s o f t h i s book. V a r i o u s h i s t o l o g i c a l a b b e r a t i o n s due t o photodynamic a c t i o n have been r e p o r t e d i n the l i t e r a t u r e , b o t h w i t h s y n t h e t i c and n a t u r a l l y

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o c c u r i n g p h o t o s e n s i t i z e r s , which r e s u l t i n p h y s i o l o g i c a l changes i n i n s e c t systems. The midgut w a l l and the crop o f the d y e - f e d , l i g h t exposed house f l y and mosquito have been observed t o s u f f e r c e l l u l a r damage. The gut was r e p o r t e d t o be d i s t e n d e d w i t h numerious a i r bubbles (24-25) s u g g e s t i n g an a l t e r a t i o n i n the membrane s t r u c t u r e which p r o b a b l y causes changes i n membrane p e r m e a b i l i t y and l y s i s o f c e l l u l a r o r g a n e l l e s i n the midgut e p i t h e l i u m . V o l u m e t r i c changes i n the haemolymph and crop c o n t e n t s o f cockroaches were observed due t o photodynamic a c t i o n (26>). These changes appear t o r e f l e c t a t r a n s f e r o f haemocoel f l u i d s i n t o the a l i m e n t a r y c a n a l and perhaps i n t o t i s s u e . The dyes may a f f e c t the p e r m e a b i l i t y o f c e l l membranes t h e r e b y c r e a t i n g a d i f f e r e n t i a l i n osmotic p r e s s u r e which a l l o w s hemocoel f l u i d s t o pass i n t o t h e a l i mentary c a n a l . A s u b s t a n t i a l decrease i n haemolymph volume over a r e l a t i v e s h o r t p e r i o d o f time may c o n t r i b u t e t o the death o f the insects. H i s t o l o g i c a l and p h y s i o l o g i c a l damage i s p r o b a b l y due t o photo dynamic a c t i o n on b i o l o g i c a l membranes and the s i n g l e t oxygen mechanism i s s u s p e c t e d i n many c a s e s . The p h y s i o l o g i c a l and h i s t o l o g i c a l e f f e c t s o f photodynamic a c t i o n have been reviewed by Weaver i n the p r e v i o u s c h a p t e r o f t h i s book. I t seems t h a t photodynamic a c t i o n a l t e r s the membrane p r o t e i n as w e l l as l i p i d components o f biomembranes ( l i p i d b i l a y e r ) . Sodium channels a r e b l o c k e d and the p e r m e a b i l i t y t o potassium i o n s i s a f f e c t e d (27-31). The a l t e r e d membrane s t r u c t u r e and changes i n the membrane p e r m e a b i l i t y may lead to c e l l death. Freeman and G i e s e (32) r e p o r t e d t h a t r o s e bengal i n i t i a l l y forms a complex a t the c e l l membrane i n y e a s t c e l l s . Illumination l e a d s t o b i n d i n g and p h o t o o x i d a t i o n , f i r s t a t the s u r f a c e and then i n the c y t o p l a s m , as t h e dye d i f f u s e s i n w a r d s . S i n g l e t oxygen passes through the c e l l membrane and d i f f u s e s i n t o the c y t o p l a s m p r o d u c t i n g damage a l o n g i t s path t o the membrane l e a d i n g t o photoh a e m o l y s i s o f the c e l l s . P o o l e r and Valenzeno (33) s t u d i e d p h o t o c h e m i c a l damage o c c u r i n g t o i n t r a c e l l u l a r components by photo s e n s i t i z i n g a g e n t s . The r o s e bengal b i n d s on the o u t e r membrane s u r f a c e w i t h i t s two n e g a t i v e charges exposed t o the aqueous medium and t h e hydrophobic p o r t i o n o f the m o l e c u l e i n s e r t e d i n the l i p i d b i l a y e r . Photodynamic l e s i o n s a r e c r e a t e d when membranebound dye m o l e c u l e s g e n e r a t e a c t i v e oxygen. P h o t o x i d a t i v e damage t o c e l l membranes l e a d s t o l e a c h i n g o f p o t a s s i u m out o f c e l l s and then t o c y t o p l a s m i c e x t r u s i o n ( 3 4 ) . The p e r m e a b i l i t y o f t h e c e l l membrane i s a l t e r e d which r e s u l t s i n m o d i f i c a t i o n o f c e l l u l a r f u n c t i o n . The l y s e d c e l l s seem t o be permeable t o e r y t h r o s i n Β ( 3 5 ) . S e v e r a l workers observed h o l e s i n the plasma membrane and m i t o c h o n d r i a appear t o be s w o l l e n and d i s t o r t e d (35-36).

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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The s w e l l i n g i s p r o b a b l y due t o the i n h i b i t i o n o f enzymatic a c t i v i t i e s i n the e l e c r o n t r a n s p o r t system and by u n c o u p l i n g o f p h o s p h o r y l a t i o n from r e s p i r a t i o n ( 3 6 ) . The photodynamic e f f e c t s o f c e r c o s p o r i n showed the changes a s s o c i a t e d w i t h l i p i d p e r o x i d a t i o n ( 3 7 ) . There was an i n c r e a s e i n the r a t i o o f s a t u r a t e d t o u n s a t u r a t e d f a t t y a c i d s , and a d e c r e a s e i n the f l u i d i t y o f the membrane r e s u l t i n g i n changes i n membrane p e r m e a b i l i t y . E l e c t r o l y t e leakage and c e l l death may be accounted f o r t h e s e p e r t u r b a t i o n s o f membrane c o m p o s i t i o n and s t r u c t u r e . Photodynamic damage i s dependent on the f a t t y a c i d c o m p o s i t i o n o f the membranes and the o s m o l a r i t y o f the medium ( 3 8 ) . I t o (39) p r o posed two modes o f c e l l - d y e i n t e r a c t i o n ; i . e . , membrane a t t a c k by e x t r a c e l l u l a r l y g e n e r a t e d s i n g l e t oxygen and a t t a c k by the dye l o c a l i z e d i n the h y d r o p h o b i c r e g i o n o f the membrane. 3.

V i t a l Enzyme System

Photodynamic a c t i o n has been observed t o cause i n a c t i v a t i o n i n s e v e r a l groups o f enzymes i n c l u d i n g the enzymes c r u c i a l t o metabo l i c pathways such as g l y c o l y s i s , the Krebs c y c l e , amino a c i d meta b o l i s m , pentose phosphate pathway, f a t t y a c i d m e t a b o l i s m and o x i d a t i v e p h o s p o r y l a t i o n (4,40-42). The v i t a l enzyme systems a f f e c t e d by photodynamic a c t i o n i n c l u d e mixed f u n c t i o n o x i d a s e s ( 4 3 ) ; cytochrome P-450 ( 4 4 ) ; a l c o h o l dehydrogenases and l i p o a m i d e dehydrogenase (45-46); glucose-6-phosphate dehydrogenase ( 4 7 ) ; c i t r a t e s y n t h e t a s e ( 4 8 ) ; ATPase and a d e n y l k i n a s e ( 4 9 ) ; a c e t y l c h o l i n e s t e r a s e (50-53); and l a c t i c dehydrogenase ( 5 4 ) . The most e x t e n s i v e l y s t u d i e d i n s e c t enzyme system w i t h photody namic a c t i o n i s the a c e t y l c h o l i n e s t e r a s e system which i s v i t a l f o r neurotransmission. I n i t i a l observation i n dye-fed, light-exposed b o l l w e e v i l s and house f l i e s showed h y p e r e x c i t a t i o n an i n c r e a s e d a c t i v i t y ( 2 4 ) . An attempt has been made t o q u a n t i t a t e the locomotary a c t i v i t y o f d y e - t r e a t e d and c o n t r o l house f l i e s u s i n g a v i b r a t i o n s e n s i t i v e a c t o g r a p h system ( T a b l e I ) . Table I .

E f f e c t o f Rose Bengal Treatment on the Locomotary A c t i v i t y o f House F l y , domestica

3

Conditions

Locomotary A c t i v i t y Control Treated

ύ

Room l i g h t 587.6>35.7 404.6>25.3 Dark 206.4>22.8 217.9>25.6 Night 13.4> 1.4 13.9> 1.9 A c t i v i t y i n u n i t s per hour f o r 25 females Mean o f 51 r e p l i c a t e s > SE S t a t i s t i c a l l y s i g n i f i c a n t a t 0.05% l e v e l

Percent D i f f e r e n c e in Activity 45.25 5.55 3.52

c

a

b

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The rose b e n g a l - t r e a t e d , l i g h t - e x p o s e d i n s e c t s showed about 45 p e r c e n t i n c r e a s e d locomotary a c t i v i t y r e l a t i v e to c o n t r o l f l i e s . The symptoms of photodynamic t o x i c i t y , such as i n c r e a s e d i r r i t a b i l i t y , i n c r e a s e d a n t e n n a l grooming, and i n c r e a s e d locomotary c o o r d i n a t i o n f o l l o w e d by p a r a l y s i s and death (24) c l e a r l y i n d i c a t e the involvement of the nervous system. S e v e r a l r e s e a r c h e r s observed the i n a c t i v a t i o n of a c e t y l c h o l i n e s t e r a s e due to photodynamic a c t i o n (24,52). I n summary, the s i n g l e t oxygen generated i n photodynamic a c t i o n i s an i n d i s c r i m i n a t e o x i d i z i n g agent such t h a t t h e r e may not be one s i n g l e c r i t i c a l t a r g e t s i t e a f f e c t e d a t one time- Death may occur i n i n s e c t s as a c u m u l a t i v e e f f e c t of the o x i d a t i o n of many d i s c r e t e targets· II.

L i g h t Independent T o x i c i t y Mechanism

The l i g h t independen i n i n s e c t s operates i n the absence o l i g h t e concentratio o t o x i c compound needed f o r the dark r e a c t i o n i s c o m p a r a t i v e l y h i g h and the time r e q u i r e d f o r the l e t h a l a c t i o n i s c o m p a r a t i v e l y l o n g e r r e l a t i v e t o the l i g h t dependent mechanism. T h i s mechanism has been observed w i t h s e v e r a l i n s e c t s p e c i e s both w i t h s y n t h e t i c dyes as w e l l as w i t h n a t u r a l p r o d u c t s . The l i g h t independent t o x i c i t y of the xanthene dyes was f i r s t i n v e s t i g a t e d by Blum (_^5). More r e c e n t l y i t has been r e p o r t e d w i t h the xanthene dyes i n f i r e ants (^6), b o l l w e e v i l s (53-54), f a c e f l i e s ( 5 7 ) , house f l i e s ( 5 8 ) , c o r n ear worms ( 5 9 ) , and mosquitoes (60), In the b e g i n n i n g i t was thought t h a t the l i g h t independent t o x i c i t y i n i n s e c t s i s due to an o r g a n o c h l o r i n e type o f t o x i c i t y (57) which r e s u l t s i n symptoms of energy s t r e s s . But the h i g h l e v e l s of dark t o x i c i t y r e p o r t e d i n the house f l i e s w i t h the nonh a l o g e n a t e d dyes such as rhodamine Β and rhodamine 6G (61) c a s t doubt on t h i s h y p o t h e s i s . The l i g h t independent t o x i c i t y w i t h n a t u r a l p r o d u c t s l i k e a l p h a t e r t l i i e n y l , phenyl h e p t a t r i e n e , and x a n t h o t o x i n was r e p o r t e d i n m o s q u i t o , b l a c k f l y , Manduca, and Spodoptera l a r v a e (62-65). The t a r g e t i n the dark r e a c t i o n w i t h the n a t u r a l p r o d u c t s appears to i n v o l v e membranes (^l,6f>) The Manduca l a r v a e fed w i t h the a l p h a t e r t h i e n y l f r e q u e n t l y produced l i q u i d f r a s s which i n d i c a t e s t h a t the h i n d gut i s f a i l i n g t o reabsorb water (65) and t h i s may be due to the d i s r u p t i o n of the e p i t h e l i a l membrane of the midgut and by i n t e r f e r e n c e w i t h the f u n c t i o n of the r e c t a l glands ( 6 7 ) .

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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The b i o c h e m i c a l changes a s s o c i a t e d w i t h the l i g h t independent t o x i c i t y w i t h t h e r o s e bengal were s t u d i e d i n t h e b o l l w e e v i l by Broome e t a l (_53). B o l l w e e v i l s f e d f o r 4 days w i t h rose bengal were 18 p e r c e n t l i g h t e r by wet weight and 41 p e r c e n t l i g h t e r by d r y weight than c o n t r o l w e e v i l s . They c o n t a i n e d 90 p e r c e n t l e s s l i p i d and 41 p e r c e n t l e s s p r o t e i n . Amino a c i d p o o l s f l u c t u a t e d d r a s t i cally. I n r e l a t e d s t u d i e s , Callaham e t a l (54) observed t h a t rose bengal f e d b o l l w e e v i l s d i d not l o s e weight o r decrease p r o t e i n l e v e l s ; r a t h e r , they remained c o n s t a n t , whereas the c o n t r o l i n s e c t s i n c r e a s e d . S t u d i e s by Waldbauer (68) a l s o i n d i c a t e d t h a t i n t r e a t e d i n s e c t s , n u t r i e n t s a r e d i v e r t e d t o r e p a i r damage and do not c o n t r i b u t e t o growth. Champaigne e t a l (£9) r e p o r t e d t h a t a l p h a t e r t h i e n y l reduces the gross e f f i c i e n c y w i t h which the d i e t i s c o n v e r t e d t o i n s e c t biomass. J o r d e n and Smith (70) suggested t h a t xanthene dyes i n h i b i t s e v e r a l w e l l known d e t o x i f i c a t i o i m p o r t a n t r o l e i n the l i g h In summary, death by the l i g h t independent t o x i c i t y i s p r o b a b l y due t o i n t e r f e r e n c e w i t h the growth and s u r v i v o r s h i p o f an i n s e c t by d i s r u p t i n g the m e t a b o l i c p r o c e s s , d i s r u p t i n g the e p i t h e l i a l membranes o f t h e g u t , by i n t e r f e r i n g w i t h n u t r i e n t a s s i m i l a t i o n o r by d e t e r r i n g f e e d i n g (69) which r e s u l t s i n a l e t h a l energy s t r e s s . III.

Developmental T o x i c i t y

D u r i n g the l a s t decade, r e s e a r c h e r s from s e v e r a l l a b o r a t o r i e s have observed and emphasized the adverse e f f e c t s o f p h o t o a c t i v e compounds on the development o f i n s e c t s . I n the developmental t o x i c i t y , e a r l i e r stages o f the i n s e c t s a r e exposed t o s u b l e t h a l doses o f the p h o t o a c t i v e compounds and t h i s r e s u l t s i n e i t h e r mort a l i t y o r some adverse e f f e c t i n a l a t e r stage o f development. These adverse e f f e c t s i n c l u d e f o r m a t i o n o f m o r p h o l o g i c a l abnorm a l i t i e s , growth r e t a r d a t i o n , p r o l o n g e d developmental p e r i o d s , u n d e r s i z e d i n d i v i d u a l s , and e f f e c t s on f e c u n d i t y , f e r t i l i t y , and the sex r a t i o i n i n s e c t s . These developmental e f f e c t s have been observed both w i t h s y n t h e t i c dyes and n a t u r a l p r o d u c t s . These e f f e c t s a r e seen i n e i t h e r the presence o r absence o f l i g h t . The c o n c e n t r a t i o n o f t h e p h o t o s e n s i t i z e r needed f o r developmental t o x i c i t y i s c o m p a r a t i v e l y low. A.

Morphological Abnormalities

S e v e r a l m o r p h o l o g i c a l and p h y s i o l o g i c a l a b n o r m a l i t i e s i n response t o treatment by p h o t o s e n s i t i z e r d u r i n g the development o f i n s e c t s have been observed. The s p e c i e s o f i n s e c t showing these morphological abnormalities include Drosophlla (71), a l f a l f a butt e r f l y , C o l i a s eurytheme (_72), mosquito (73-75), f a c e f l y ( 7 6 ) ; house f l y ( P i m p r i k a r , u n p u b l i s h e d ) ; P a p i l i o b u t t e r f l y ( 7 7 ) ,

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tobacco horn worm, Manduca s e x t a ( 6 9 , 2 8 ) , and unpublished).

f i r e ant

(Pimprikar,

The l e v e l o f m o r p h o l o g i c a l a b n o r m a l i t i e s induced i s dependent on v a r i o u s f a c t o r s such as c o n c e n t r a t i o n o f the s e n s i t i z e r l e n g t h of exposure, presence of l i g h t , stage of i n s e c t , mode o f a p p l i c a t i o n of the s e n s i t i z e r , and the s p e c i e s of i n s e c t used i n the experiment. Many of the m o r p h o l o g i c a l a b n o r m a l i t i e s resemble the e f f e c t s induced by the j u v e n i l e hormone analogs or the c h i t i n s y n t h e s i s i n h i b i t o r such as D i m i l i n . D u r i n g the l a r v a l p e r i o d , many of the i n s e c t s s u r v i v e the dye treatment at lower c o n c e n t r a t i o n s and remain o u t w a r d l y u n a f f e c t e d u n t i l molting begins. V a r i o u s m o r p h o l o g i c a l a b n o r m a l i t i e s observed i n house f l i e s , mosquitoes, face f l i e s and f i r e ants are shown i n F i g u r e 1. I n the case of hous p o s t e r i o r regions e x h i b i t e pupatio regio remained as l a r v a e ( F i g . 1A). These i n d i v i d u a l s c o u l d not s u r v i v e beyond p u p a t i o n and d i e d i n t h a t s t a g e . I n the case of m o s q u i t o e s , the t r e a t e d l a r v a e were unable t o shed the o l d c u t i c l e from the abdomen and head r e g i o n . The p a r t i a l l y shed exuvium remained a t t a c h e d t o the l a r v a e . Some l a r v a e s t r u g g l e d l a b o r i o u s l y t o shed the e x u v i e but f a i l e d and e v e n t u a l l y d i e d i n the p r o c e s s ( F i g . I B ) . There were s e v e r a l m o r p h o l o g i c a l i n t e r m e d i a t e s observed w i t h pupal head c a p s u l e and l a r v a l abdominal segments. Some pupae r e t a i n e d the 4 t h i n s t a r c u t i c l e but those t h a t pupated s u c c e s s f u l l y o f t e n d i e d l a t e r . F a i l u r e of proper a d u l t e c l o s i o n i s the most p r e v e l e n t o f a l l the e f f e c t s n o t e d . The f a i l u r e of a d u l t s t o emerge c o m p l e t e l y from the puparium v a r i e d from complete l a c k of e c l o s i o n t o o n l y s l i g h t attachment of the wing or l e g t o the puparium ( F i g . 1C). In the m a j o r i t y of c a s e s , o n l y the head emerged from the puparium. I n o t h e r c a s e s , the emerging a d u l t was s u c c e s s f u l i n s e p a r a t i n g body p a r t s up to the t h o r a x or even the l e g s and h a l f of the abdomen from the pupal exuvium. Sometimes, the a d u l t e s s e n t i a l l y comes out of the puparium but i s s t i l l a t t a c h e d by v a r i o u s appendages and cannot f r e e i t s e l f c o m p l e t e l y . I n many i n s t a n c e s s u c c e s s f u l l y emerged a d u l t s are not as h e a l t h y or a c t i v e . Many of them appear t o be s m a l l i n s i z e ( 7 9 ) . The wings of s u c c e s s f u l l y emerged a d u l t s may be c u r l e d , s h o r t , and n o n - f u n c t i o n a l ( F i g . ID, F i g . IE) as seen i n the mosquito ( 7 5 ) , face f l y ( 7 6 ) , f i r e ant and house f l y ( P i m p r i k a r , u n p u b l i s h e d ) . M o r p h o l o g i c a l l y normal face f l i e s which emerged from e r y t h r o e i n B - t r e a t e d manure were shown t o have a s h o r t e r l i f e span than those emerged from c o n t r o l manure ( 7 9 ) . T h i s t o x i c i t y i s

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F i g u r e 1. V a r i o u s m o r p h o l o g i c a l a b n o r m a l i t i e s observed due t o the dye treatment i n (A) house f l y , (B) mosquito, (C) house f l y , (D) deformed wings i n face f l y , and (E) d e f o r m i t i e s i n wing i n f i r e ant s.

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p r o b a b l y due t o the e f f e c t s o f the r e s i d u a l dye l e v e l s consumed i n the l a r v a l stage and m a i n t a i n e d i n t h e t i s s u e through the p u p a l stage i n t o t h e a d u l t s t a g e . Q u a n t i t a t i v e s t u d i e s on the e f f e c t o f s e n s i t i z e r s on a d u l t emergence and a l s o on m o r p h o l o g i c a l a b n o r m a l i t i e s were c a r r i e d out i n mosquitoes ( 7^), face f l i e s (79), P i m p r i k a r ( u n p u b l i s h e d ) . The d a t a i n Table I I i n d i c a t e s t h a t the a b n o r m a l i t i e s as w e l l as a d u l t emergence i s dependent on t h e c o n c e n t r a t i o n o f t h e p h o t o s e n s i t i z e r . Table I I .

E f f e c t o f Rose Bengal and E r y t h r o s i n Β on House F l y Development 3

Treatment

Control 22 ppm 44 ppm 110 ppm

Percent Reduction i n A d u l t Emergence^ RB EB

-

26.4 40.3 64.0

P e r c e n t Abnormal Pupae RB EB c

3.6 14.7 21.6

22.6 39.5 54.2

3.8 23.7 21.6

a

Average of three r e p l i c a t e s ^ P e r c e n t r e d u c t i o n compared t o c o n t r o l C o r r e c t e d f o r c o n t r o l by A b b o t t s f o r m u l a c

1

V a r i o u s r e s e a r c h e r s attempted t o e x p l a i n these m o r p h o l o g i c a l abnormalities. Many o f these problems seem t o be a s s o c i a t e d w i t h normal muscle attachment. I t seems t h a t the enhanced m o r t a l i t y , as w e l l as a b o r t i v e m o l t i n g , may be due t o the e f f e c t s o f t h e e x e r t i o n r e q u i r e d a t the emergence on a weakened i n s e c t . The treatment o f the p h o t o s e n s i t i z e r s r e s u l t s i n a decrease i n the weight o f the i n s e c t , r e d u c t i o n i n t o t a l l i p i d and p r o t e i n c o n t e n t s (53-54). The p h o t o s e n s i t i z e r s a r e a l s o c a p a b l e o f c a u s i n g s e v e r a l b i o c h e m i c a l changes i n the i n s e c t system which c o u l d l e a d t o s t r e s s f u l development o f an i n d i v i d u a l . These weakened i n s e c t s p r o b a b l y can not r e s i s t muscular t e n s i o n and i n c r e a s e d t u r g o r p r e s s u r e d u r i n g the p r o c e s s o f m o l t i n g which may r e s u l t i n a b o r t i v e m o l t i n g . Champaigne e t a l (69) r e p o r t e d t h a t the p a r t i a l l y molted c u t i c l e c o n s t r i c t s t h e l a r v a e o f M^ s e x t a when fed w i t h t h e photo s e n s i t i z e r . T h i s p r e v e n t s the passage o f the gut c o n t e n t s and r e s t r i c t s the c i r c u l a t i o n o f t h e haemolymph. E v e n t u a l l y , t h e a n t e r i o r p a r t o f t h e l a r v a e becomes t u r g i d and t h e l a r v a e stops f e e d i n g and f i n a l l y d i e s . Downum e t a l (_78) observed t h a t t h e a b n o r m a l i t i e s i n M^ s e x t a l a r v a e caused by i n g e s t i o n o f a l p h a t e r t h i e n y l a r e s i m i l a r t o t h e a b n o r m a l i t i e s caused by t h e a c t i o n o f L-dopa i n t h e s o u t h e r n army worm as r e p o r t e d by Rehr e t a l (£0). A c c o r d i n g t o Rehr, the deformed p u p a t i o n might be due t o the i n t e r f e r e n c e o f t y r o s i n a s e ,

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which i s an e s s e n t i a l enzyme f o r h a r d e n i n g and d a r k e n i n g of the c u t i c l e , by the n o n - p r o t e i n amino a c i d s . Downum et a l (31) recorded i n c o l i t h a t the s i n g l e t oxygen, produced by UV-A a c t i v a t e d a l p h a t e r t h i e n y l , c r o s s - l i n k s the membrane p r o t e i n s . They s p e c u l a t e t h a t s i m i l a r e f f e c t s caused by s i n g l e t oxygen i n the integument of s e x t a might be r e s p o n s i b l e f o r the d e f o r m i t i e s i n sclerotization. Most a b n o r m a l i t i e s appear t o be a s s o c i a t e d w i t h problems i n m o l t i n g which l e a d t o the h y p o t h e s i s t h a t these p h o t o s e n s i t i z e r s may have an e f f e c t on the m o l t i n g hormones. The two most prominant m o l t i n g hormones i n i n s e c t s are alpha-ecdysone ( e c d y s t e r o n e ) and beta-ecdysone ( 2 0 - h y d r o x y e c d y s t e r o n e ) . These hormones are s t e r o i d a l i n n a t u r e and the t i t e r s of these hormones c o n t r o l the sequence o f developmental events such as m o l t i n g , p u p a t i o n , a d u l t development and o o g e n i s i s ( 8 1 ) . An HPLC procedure f o two s t e r o i d hormones was r e p o r t e d by P i m p r i k a r et a l ( 8 2 ) . The t i t e r s of ecdysterone and 20-hydroxyecdysterone d u r i n g the d e v e l o p ment of the c o n t r o l and e r y t h r o s i n B - t r e a t e d house f l i e s are shown i n F i g u r e 2A and F i g u r e 2B. The t i t e r s of the hormones as w e l l as the r a t i o of a l p h a - and beta-ecdysones are d i s t i n c t l y d i f f e r e n t i n the e r y t h r o s i n B - t r e a t e d i n s e c t s as compared to the c o n t r o l i n s e c t s . I t i s thought t h a t the imbalance o f the m o l t i n g hormone t i t e r s d u r i n g the c r i t i c a l stages o f development may c o n t r i b u t e t o the a b o r t i v e m o l t i n g or t o the development of m o r p h o l o g i c a l l y abnormal i n d i v i d u a l s . An important f a c t o r which needs f u r t h e r c o n s i d e r a t i o n i s the o b s e r v a t i o n t h a t some l a r v a e s u c c e s s f u l l y pupated and of these some s u c c e s s f u l l y emerged as abnormal or normal a d u l t s . T h i s might be due to an i n a b i l i t y t o s e l e c t l a r v a e f o r treatment w i t h the photos e n s i t i z e r which were i n c o m p l e t e l y synchronous development. I t a l s o suggests t h a t t h e r e are s p e c i f i c "developmental time windows" o n l y through which the p h o t o s e n s i t i z e r can be e f f e c t i v e l y i n t r o duced to cause morphogenetic e f f e c t s . B.

Delayed Developmental P e r i o d s

Other developmental t o x i c i t y e f f e c t s of the p h o t o s e n s i t i z e r s are r e f l e c t e d by the s i g n i f i c a n t d e l a y s i n developmental p e r i o d s i n i n s e c t s . Two i n t e r r e l a t e d areas of i n t e r e s t w i t h the d e l a y e d developmental p e r i o d i n c l u d e the a n t i f e e d a n t a c t i v i t y of the photosens i t i z e r s and the development o f s m a l l e r s i z e d i n d i v i d u a l s . E a r l y r e s e a r c h by Edwards (8J3) r e p o r t e d the r e t a r d a t i o n o f growth i n s i l k w o r m l a r v a e f e d on l e a v e s s p r i n k l e d w i t h methylene b l u e . I n t h i s i n s t a n c e , the author suggested t h a t the low p a l a t a b i l i t y of the dyed l e a v e s may have caused the r e t a r d a t i o n o f l a r v a l growth. K o y l e r (72) r e p o r t e d t h a t the growth of the a l f a l f a c a t e r p i l l a r , C o l i a s p h i l o d i c e and eurytheme, was prolonged when

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

F i g u r e 2 . T i t e r s of (A) alpha-ecdysone and (B) beta-ecdysone d u r i n g the development of house f l y .

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exposed t o n e u t r a l r e d . D a v i d (71) observed t h a t methylene b l u e can r e t a r d the growth o f D r o s o p h l l a l a r v a e and t h a t the r e t a r d a t i o n ranged from 17 t o 400 hours w i t h i n c r e a s i n g c o n c e n t r a t i o n o f dye. Barbosa and P e t e r (84) demonstrated i n a s e r i e s of e x p e r i ments, r e t a r d a t i o n of growth i n the l a r v a e of the mosquito, Aedes a e g y p t i exposed t o methylene b l u e and n e u t r a l r e d . The number o f hours r e q u i r e d f o r 50 percent or more l a r v a e t o pupate i n c r e a s e s d r a m a t i c a l l y . The r e t a r d a t i o n i n some cases was a p p r o x i m a t e l y 10 times t h a t of c o n t r o l . In a l l the experiments a t t e m p t i n g t o i l l u s t r a t e the r e t a r d a t i o n of growth, they used the f o l l o w i n g t h r e e criteria: 1.

2. 3.

Delay i n onset of p u p a t i o n Length of l a r v a l p e r i o d R e l a t i v e rates of pupation

The d e l a y i n the p e r i o dent. The e f f e c t of exposure seemed t o be l e s s severe on the l a t e r i n s t a r s . The a u t h o r s concluded t h a t the l e n g t h and the stage of exposure may have a key r o l e i n the e f f e c t s o f dyes. They a l s o conducted experiments to determine i f t h e r e were any d i f f e r e n c e s i n the amount o f food ( y e a s t s u s p e n s i o n ) t h a t the mosquito l a r v a e would i n g e s t when p l a c e d i n v a r i o u s c o n c e n t r a t i o n s of dye. The main r e a s o n f o r t h i s experiment was the p o s s i b i l i t y t h a t r e t a r d a t i o n o f growth might have been caused s i m p l y by l a c k of f e e d i n g due to u n p a l a t a b l e food. There was no s i g n i f i c a n t d i f f e r e n c e i n average l a r v a l w e i g h t s i n d i c a t i n g t h a t the r e t a r d a t i o n of growth was not caused by r e j e c t i o n of dyed food under the c o n d i t i o n s of the experiment. Clement et a l (&5) a l s o observed the r e t a r d e d l a r v a l growth i n the b l a c k c u t worm. However, t h e r e was a remarkable decrease i n the number o f f e c a l p e l l e t s i n the d y e - t r e a t e d l a r v a e which i n d i c a t e d t h a t the l a r v a e consumed r e l a t i v e l y s m a l l e r amounts of dyet r e a t e d food. Q u a n t i t a v e s t u d i e s on the delayed developmental p e r i o d s i n the house f l y due t o e r y t h r o s i n Β and rose b e n g a l t r e a t ment were conducted i n our l a b o r a t o r y . The data i n the T a b l e I I I i n d i c a t e s t h a t the l a r v a l and pupal p e r i o d s were p r o l o n g e d which were u l t i m a t e l y r e f l e c t e d i n a c o r r e s p o n d i n g d e l a y i n a d u l t house f l y emergence. There was a d e l a y of about 3 t o 4 days i n the a d u l t emergence i n house f l i e s r e a r e d on the d y e - t r e a t e d medium and the developmental d e l a y was dependent on the c o n c e n t r a t i o n o f the dye (Table I I I ) .

American Chemical Society, Library 1155 16th St.,

N.W.

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Table I I I .

Emergence on Day 1 2 3 4 5 3

Delayed A d u l t Emergence Due t o E r y t h r o s i n Β Treatment i n House F l y 3

Cumulative p e r c e n t A d u l t Emergence 110PPM 22PPM Control 33.5 75.2 95.3 97.7 99.9

7.1 37.4 87.9 94.2 99.9

0.7 20.6 52.9 89.4 99.9

Average of three r e p l i c a t e s

The d e l a y e d developmental e f f e c t s o f the p h o t o s e n s i t i z e r s have been s t u d i e d r e c e n t l y b (77) s t u d i e d the t o x i c i t and a n g u l a r furanocoumarins on the P a p i l i o b u t t e r f l i e s . The l a r v a e fed on l e a v e s c o n t a i n i n g a n g e l i c i n grew more s l o w l y and weighed l e s s a t p u p a t i o n . The a u t h o r s c o r r e l a t e d the reduced pupal w e i g h t s w i t h the reduced a d u l t body s i z e and concluded t h a t the d e l e t e r i o u s e f f e c t i s due to i n g e s t i o n o f a n g e l i c i n and not due t o reduced consumption. Downum et a l (78) a d m i n i s t e r e d a l p h a t e r t h i e n y l t o the tobacco horn worm, through an a r t i f i c i a l d i e t and observed a d e l a y i n p u p a t i o n o f the l a r v a e . S i m i l a r l y Kagan e t a l (86) a l s o r e p o r t e d p r o l o n g e d l a r v a l p e r i o d s i n the mosquito due t o alpha t e r t h i e n y l treatment. A l p h a t e r t h i e n y l and phenyl h e p t a t r i y n e are known t o be p o t e n t f e e d i n g i n h i b i t o r s i n s e v e r a l i n s e c t s p e c i e s l i k e the European c o r n b o r e r , the cut worm, the tobacco budworm, and the Colorado p o t a t o b e e t l e (69,87-88). The l a r v a e o f M. s e x t a consumed l i t t l e d i e t and produced few f e c a l p e l l e t s and i t was suggested t h a t s t a r v a t i o n c o n t r i b u t e d t o m o r t a l i t y (69). T h e i r s t u d i e s a l s o demonstrated t h a t photodynamic p l a n t p r o d u c t s can l e n g h t e n l a r v a l development t i m e , reduce growth, decrease the e f f i c i e n c y of c o n v e r s i o n o f i n g e s t e d f o o d , and the e f f i c i e n c y o f c o n v e r s i o n o f d i g e s t e d f o o d . A n t i f e e d e n t a c t i v i t y experiments c l e a r l y i n d i c a t e d t h a t a l p h a t e r t h i e n y l reduces f e e d i n g a c t i v i t y . The net e f f e c t o f the a n t i f e e d e n t a c t i v i t y o f p h o t o s e n s i t i z e r s p r o b a b l y r e s u l t s i n the development o f s m a l l e r s i z e d i n d i v i d u a l s as demonstrated by D a v i d (71) i n D r o s o p h l l a , K o y l e r (72) i n the a l f a l f a c a t e r p i l l a r and by Berenbaum et a l (77) i n P a p i l i o butterflies. Barbosa and P e t e r s (84) observed t h a t female pupal w e i g h t s i n A. a e g y p t i decreased s i g n i f i c a n t l y due t o the treatment of photo s e n s i t i z e r . However, male pupal w e i g h t s were not a f f e c t e d . They proved e x p e r i m e n t a l l y t h a t t h i s was not caused by r e j e c t i o n o f dyeimpregnated f o o d . S a k u r a i and H e i t z (89) r e p o r t e d decreased pupal w e i g h t s i n the r o s e b e n g a l - and e r y t h r o s i n B - t r e a t e d house f l i e s .

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149

The l a r v a e o f the c u t worm, Euxoa m e s s o r i a showed s i g n i f i c a n t l y depressed growth due t o a l p h a t e r t h i e n y l f e e d i n g . The l a r v a l and pupal weights were decreased by about 30 p e r c e n t ( 6 9 ) . The d e l a y e d developmental p e r i o d s and the a n t i f e e d e n t p r o p e r t i e s o f the p h o t o s e n s i t i z e r s w i t h the r e s u l t i n g e f f e c t s on r e t a r d a t i o n o f growth have profound i m p l i c a t i o n s i n the p r a c t i c a l a p p l i c a t i o n o f the p h o t o s e n s i t i z e r s i n i n t e g r a t e d pest c o n t r o l programs i n the f o l l o w i n g ways: 1.

The number of g e n e r a t i o n s per season c o u l d be reduced due to the p r o l o n g e d growth p e r i o d s .

2.

S i n c e the l a r v a l and pupal p e r i o d s take l o n g e r f o r development, i t g i v e s a d d i t i o n a l time f o r p a r a s i t e s , p r e d a t o r s , and n a t u r a l enemies f o r effective contro harmless pupal s t a g e s )

3.

The growth r e t a r d e d i n d i v i d u a l s are l i k e l y t o experience a s u b s t a n t i a l reduction i n f i t n e s s compared to the normal i n s e c t s .

A c c o r d i n g t o Lewontin ( 9 0 ) , even s m a l l changes i n the development time can have g r e a t e f f e c t s on r e p r o d u c t i v e p o t e n t i a l . There i s a need f o r f u r t h e r r e s e a r c h on the mechanisms by which the r e t a r d a t i o n o c c u r s so t h a t i t can be more p r e c i s e l y e x p l o i t e d f o r insect control. C.

B i o t i c , O v i c i d a l , and Other

Effects

D u r i n g the l a s t decade, s t u d i e s have i n d i c a t e d t h a t n a t u r a l l y o c c u r i n g and s y n t h e t i c p h o t o s e n s i t i z e r s are both capable o f c a u s i n g d e l e t e r i o u s b i o t i c e f f e c t s i n i n s e c t s . T h i s i n c l u d e s e f f e c t s on f e c u n d i t y and f e r t i l i t y . F e c u n d i t y r e p r e s e n t s the number of eggs l a i d by the female over her e n t i r e l i f e t i m e and f e r t i l i t y r e p r e s e n t s the v i a b i l i t y o f the l a i d eggs by the females. D a v i d (71,91) f o r the f i r s t time observed t h a t f e c u n d i t y i n D r o s o p h l l a was markedly lower due to the methylene b l u e t r e a t m e n t . P i m p r i k a r et a l (92!) demonstrated the e f f e c t of rose bengal on f e c u n d i t y and f e r t i l i t y i n the house f l y . F e c u n d i t y was observed to be reduced by 26 t o 69 p e r c e n t due to dye t r e a t m e n t . A r e d u c t i o n of house f l y f e c u n d i t y was observed to be d i r e c t l y r e l a t e d t o the d i e t a r y c o n c e n t r a t i o n o f the r o s e b e n g a l and the frequency o f f e e d i n g . Even though t h e r e was no remarkable e f f e c t on the v i a b i l i t y o f the eggs, t h e r e seemed t o be a p p r o x i m a t e l y a 5 t o 26 p e r c e n t r e d u c t i o n i n the v i a b i l i t y o f eggs l a i d by the female house f l i e s which were f e d on r o s e bengal (92)· There was no s i g n i f i c a n t change i n

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the sex r a t i o due to the p h o t o s e n s i t i z e r s i n mosquitoes (73) house f l i e s ( P i m p r i k a r , u n p u b l i s h e d )

and

Barenbaum and Freeny (77) w h i l e s t u d y i n g the e f f e c t of the furanocoumarins i n P a p i l i o , observed t h a t t h e r e was a 3- t o 5 - f o l d d i f f e r e n c e i n the average egg p r o d u c t i o n between the c o n t r o l and a n g e l i c i n treatments. The i n d i v i d u a l b u t t e r f l i e s i n the c o n t r o l t r e a t m e n t s l a i d up to 700 more eggs than the t r e a t e d females. The a u t h o r s c o r r e l a t e d the reduced pupal w e i g h t s w i t h the reduced body s i z e which a l s o c o r r e l a t e s w i t h f e c u n d i t y . The assumption here i s t h a t the i n s e c t s which are developed on the medium t r e a t e d w i t h the p h o t o s e n s i t i z e r produce a b n o r m a l l y s m a l l e r and l i g h t e r weight i n d i v i d u a l s and these a d u l t s are not c a p a b l e of p r o d u c i n t a normal complement of eggs. I t has been observed t h a t a l l the l i f e stages o f i n s e c t s are s u s c e p t i b l e to the a c t i o are c a p a b l e of c a u s i n g t o x i c i t When house f l y eggs are t r e a t e d w i t h p h o t o s e n s i t i z e r s and exposed to l i g h t , s e v e r a l of the xanthene dyes e x h i b i t e d o v i c i d a l a c t i v i t y ( 9 2 ) . The r e l a t i v e l y f l a t s l o p e s of the l o g dose v e r s u s p r o b i t m o r t a l i t y l i n e s i n d i c a t e t h a t the r a t e s of p e n e t r a t i o n of the pho t o s e n s i t i z e r s through the c h o r i o n i s v e r y slow or the eggs are not as s e n s i t i v e to dyes as the o t h e r l i f e s t a g e s . Some of the t r e a t e d house f l y eggs t o t a l l y f a i l t o h a t c h p r o b a b l y due to the death o f the embryo. I n some c a s e s , the l a r v a e f r e e themselves from the head c a p s u l e , but the caudal end s t i l l remains i n the egg s h e l l . V a r i o u s o t h e r a b n o r m a l i t i e s i n the h a t c h i n g of the eggs were o b s e r v e d . E o s i n Y and P h l o x i n Β treatment caused p i t t i n g of egg c e l l membranes, v a c u o l e f o r m a t i o n , and e v e n t u a l d i s i n t e g r a t i o n i n sea u r c h i n eggs ( 9 3 ) . Kagan and Chan (94) r e p o r t e d the o v i c i d a l e f f e c t s of some of the photodynamic n a t u r a l p r o d u c t s i n melanog a s t e r and suggested t h a t the p h o t o s e n s i t i z e d enhancement of the o v i c i d a l a c t i v i t y can be a p p r e c i a b l y i n c r e a s e d by p r o p e r l y s e l e c t i n g the i r r a d i a t i o n p e r i o d . Q u a n t i t a t i v e s t u d i e s on the e f f e c t s of p h o t o s e n s i t i z e r t r e a t ment a t v a r i o u s l a r v a l and pupal stages on a d u l t emergence were conducted i n face f l i e s (76) and house f l i e s ( 9 2 ) . F i g u r e 3 sum m a r i z e s the e f f e c t s o f the r o s e bengal treatment a t each stage of development i n the house f l y . The dye t r e a t e d female house f l i e s produce r e l a t i v e l y fewer eggs and these eggs are c o m p a r a t i v e l y l e s s v i a b l e . The dye t r e a t e d eggs show o v i c i d a l a c t i v i t y r e s u l t i n g i n r e d u c t i o n i n the egg hatch. The l a r v a e r e a r e d on the medium c o n t a i n i n g the photosen s i t i z e r s e x h i b i t i n c r e a s e d m o r t a l i t y p r i o r t o p u p a t i o n and r e s u l t e d i n up t o 80 p e r c e n t r e d u c t i o n i n a d u l t emergence depending on the stage of exposure and the c o n c e n t r a t i o n of the p h o t o s e n s i t i z e r (Fig. 3).

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

9. PIMPRIKAR AND COIGN

Multiple Mechanisms of Dye-Induced Toxicity

F i g u r e 3. The developmental e f f e c t s of rose bengal on v a r i o u s stages of house f l y .

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These s t u d i e s add t o the concept t h a t the p h o t o s e n s i t i z e r s a r e c a p a b l e o f c a u s i n g t o x i c i t y from the egg t o the a d u l t stage and t h a t the e f f e c t s are complex. I t i s v e r y d i f f i c u l t t o a n a l y z e t h e e f f e c t s a t each stage which a l s o suggests t h a t the e v e n t u a l f i e l d e f f e c t i v e n e s s would be d i f f i c u l t t o e s t i m a t e based on a study o f t o x i c i t y at a s i n g l e l i f e stage. In c o n c l u s i o n , t h e r e are s e v e r a l t o x i c mechanisms i n o p e r a t i o n at a g i v e n time i n a d d i t i o n t o the l i g h t dependent t o x i c mechanism. I t i s v e r y d i f f i c u l t t o i s o l a t e o r d e f i n e the r e l a t i v e c o n t r i b u t i o n of each o f these mechanisms a t a g i v e n t i m e . Acknowledgments T h i s work was supported i n f u l l by the M i s s i s s i p p i A g r i c u l t u r a l and F o r e s t r y Experiment S t a t i o n . The a u t h o r would l i k e t o thank M i s s Mary Jane Coign f o m a n u s c r i p t and Mrs. Debbi m a n u s c r i p t . MAFES p u b l i c a t i o

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78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94.

Multiple Mechanisms of Dye-Induced Toxicity

Downum, K.R.; Rosenthal, G.A.; Towers, G.H.N. Pest. Biochem. Physiol. 1984, 22, 104-109. Fairbrother, T . E . ; Essig, H.W.; Combs, R . L . ; Heitz, J.R. Environ. Entomol. 1981, 10, 506-510. Rehr, S.S.; Janzen, D.H.; Feeny, P.P. Science, 1973, 181, 81-82. Hsiao, T . H . ; Hsiao, C. J. Insect Physiol. 1977, 23, 89-93. Pimprikar, G.D.; Coign, M . J . ; Sakurai, H . ; Heitz, J.R. J . Chrom. 1984, 317, 413-419. Edwards, W.F. Text. World. 1921, 60, 1111-1113. Barbosa, P.; Peters, T.M. J. Med. Entomol. 1970, 7, 693-696. Clement, S . L . ; Schmidt, R.S.; Szatmari-Goodman, G . ; and Levine, E. J. Econ. Entomol. 1980, 73, 390-392. Kagan, J.; Hasson, M.; Grynspan, F. Biochim. Biophys. Acta, 1984, 802, 442-447. McLachlan, D.; Arnason, J . T . ; Philogene, B.J.R.; Champagne, D. Experientia, 1982 Jermy, T . ; Butts, B.A. 1, 237-242. Sakurai, H . ; Heitz, J.R. Environ. Entomol. 1982, 11, 467-470. Lewontin, R.C. In The Genetics of Colonizing Species; Baker, H.G.; Stebbine, G . L . , Eds.; Academic Press, New York, 1965; pp.588. David, J. C.R. Acad. Sci. Paris. 1955, 241, 116-118. Pimprikar, G.D.; Noe, B . L . ; Norment, B.R.; Heitz, J.R. Environ. Entomol. 1980, 9, 785-788. Tennent, D.H. In Papers From Tortugas Laboratory Vol. XXXY; Carnegie Institute of Washington, Washington, D.C. 1942; Publication #539. Kagan, J.; Chan, G. Experientia, 1983, 39, 402-403.

RECEIVED February 11, 1987

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 10

Field Development of Photooxidative Dyes as Insecticides 1

2

1

3

3

Lisa A. Lemke , P. G. Koehler , R. S. Patterson , Mary B. Feger , and Thomas Eickhoff 1

Insects Affecting Man and Animals Research Laboratory (IAMARL), Agricultural Research Service, U.S. Department of Agriculture, Gainesville, FL 32604 Department of Entomology and Nematology, University of Florida, Gainesville, FL 32611 Hilton Davis Chemical Company, 2235 Langdon Farm Road, Cincinnati, OH 45237 2

3

Erythrosin Β (Synerid) a photooxidative dye has been shown to have insecticidal properties against adult house f l i e s in small scale poultry tests conducted in FL. It provided up to 95% reduction of the adult house fly population in one of these tests. It is not, however, commercially satisfac tory as a house fly larvicide. Erythrosin B, acridine red, and rose bengal have all been used experimentally to control mosquito larvae in small pools. Erythrosin Β also shows promise as a single mound treatment for control of red imported fire ants (RIFA). It controlled RIFA colonies as effectively as Amdro through 56 day post -treatment. The photooxidative dyes are extremely safe to man and the environment. Erythrosin Β has an L D 50 of 6,000-7,000

mg/kg of body weight. S i n c e t h i s i s a symposium on l i g h t - a c t i v a t e d p e s t i c i d e s , i t o n l y seems r i g h t t h a t t h e f i e l d e v a l u a t i o n and c o m m e r c i a l d e v e l o p m e n t o f t h e s e compounds be e x a m i n e d . The t r u e t e s t f o r any p e s t i c i d e i s how i t p e r f o r m s under actual f i e l d conditions. V a r i o u s s t u d i e s have shown t h a t a number o f i n s e c t s p e c i e s e x h i b i t p h o t o o x i d a t i v e t o x i c r e a c t i o n s when exposed t o c e r t a i n dyes. Dyes s u c h as e r y t h r o s i n Β and rose bengal a r e e f f e c t i v e c o n t r o l agents a g a i n s t the a d u l t s t a g e o f t h e house f l y ( 1 - 4 ) , f a c e f l y {5), b l a c k i m p o r t e d f i r e a n t { § ) , and b o l l w e e v i l ( 7 - 8 ) . Toxic r e a c t i o n s t o these dyes i n the l a r v a l stage o f m o s q u i t o e s ( 9 - 1 1 ) , house and f a c e f l i e s ( 1 2 - 1 4 ) , y e l l o w mealworms ( 1 5 ) , cabbage b u t t e r f l i e s ( 1 6 ) , and b l a c k cutworms (17) have been o b s e r v e d i n t h e l a b o r a t o r y . 0097-6156/87/0339-0156$06.00/0 © 1987 American Chemical Society

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Photooxidative Dyes as Insecticides

D e s p i t e the promising i n d i c a t i o n s of these e a r l y l a b o r a t o r y e x p e r i m e n t s , l i t t l e work has been c o n d u c t e d t o e v a l u a t e t h e i n s e c t i c i d a l a c t i v i t y o f t h e dyes i n t h e field. Most o f t h e f i e l d work so f a r i n v o l v e d t e s t s i n p o u l t r y f a c i l i t i e s f o r t h e c o n t r o l o f house f l i e s u s i n g e r y t h r o s i n Β under t h e name S y n e r i d F l y C o n t r o l B. A t p r e s e n t , t h i s i s t h e o n l y dye r e g i s t e r e d and commercially a v a i l a b l e f o r i n s e c t c o n t r o l . This product i s l a b e l l e d f o r c o n t r o l o f house f l i e s i n c o n f i n e d animal areas, i n c l u d i n g p o u l t r y f a c i l i t i e s . One o f t h e g r e a t e s t a d v a n t a g e s o f p h o t o o x i d a t i v e d y e s i s t h e i r low mammalian t o x i c i t y ( T a b l e I ) . E r y t h r o s i n Β i s r e l a t i v e l y h a r m l e s s t o mammals (18-19) and has an a c u t e o r a l L D 5 0 o f 6,700-7,000 mg/kg o f body weight i n r a t s (19). A number o f o t h e r d y e s a l s o show low mammalian t o x i c i t y when compared t o commonly used i n s e c t i c i d e s (Tabl mammalian t o x i c i t y t h e e n v i r o n m e n t (22) and t h e r e i s l i t t l e t h r e a t o f c o n t a m i n a t i n g water s o u r c e s o r a c c u m u l a t i n g i n t h e f o o d chain. The p h o t o o x i d a t i v e d y e s a r e e x t r e m e l y s a f e and p o s e no t h r e a t t o t h e h e a l t h o r w e l f a r e o f t h e a p p l i c a t o r or environment i n f i e l d usage.

Table

I.

The L D 5 0 f o r V a r i o u s Dyes Which Show Insecticidal Properties. 3

Compound A r t i e w h i t e Tx D&C r e d #22 FD&C b l u e #02 FD&C r e d #03 FD&C y e l l o w #06 Methylene blue Sodium f l u o r e s c e i n Erythrosin Β Eosin yellow

Acute O r a l 16,000 2,344 A

1,264 12,750 1,180* 6,721* 6,700* 2,340

1

Intravenous

1

550 93* 700

82

-

—

a

R T E C S , N i o s h a , S u p t . o f Documents, U.S. Government P r i n t O f f i c e : W a s h i n g t o n , DC, 1983. T h i s f i g u r e r e p r e s e n t s mg/kg o f body w e i g h t ( f o r m i c e ) r e q u i r e d t o k i l l 50% o f t h e t e s t population. * R a t s were u s e d as t e s t o r g a n i s m . 1

House F l y F i e l d

Experiments

Larvicide Tests. F i e l d t e s t s were n e c e s s a r y t o s a t i s f y EPA r e q u i r e m e n t s f o r r e g i s t r a t i o n . Poultry f a c i l i t i e s o f f e r a p e r f e c t environment f o r t e s t i n g products a g a i n s t house f l i e s s i n c e t h e y a r e i d e a l f l y - b r e e d i n g h a b i t a t s .

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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T h i s i s b e c a u s e i n most caged l a y e r o p e r a t i o n s the manure i s a l l o w e d t o a c c u m u l a t e under c a g e d hens p r o v i d i n g e x c e l l e n t o v i p o s i t i o n and l a r v a l d e v e l o p m e n t s i t e s (23). Unchecked f l y d e v e l o p m e n t r e s u l t s i n a d u l t f l i e s creating a nuisance. Since house f l i e s a r e d e v e l o p i n g r e s i s t a n c e t o many o f the c u r r e n t l y u s e d p r o d u c t s l i k e t h e s y n t h e t i c p y r e t h r o i d s and c y r o m a z i n e ( 2 4 - 2 5 ) , the poultry i n d u s t r y needs a l t e r n a t i v e s f o r house f l y c o n t r o l . The new, s a f e r p h o t o o x i d a t i v e dyes may f i l l this requirement. One o f t h e e a r l i e s t f i e l d t e s t s u s i n g S y n e r i d was c o n d u c t e d a t the M i s s i s s i p p i S t a t e U n i v e r s i t y to c o n t r o l house f l i e s i n l a r g e and s m a l l s c a l e f i e l d t e s t s (26). In b o t h s t u d i e s , manure was s p r a y e d w e e k l y w i t h an aqueous s o l u t i o n o f e r y t h r o s i n Β f o r 4 weeks. Manure samples were c o l l e c t e populations, while s t i c k a d u l t house f l y p o p u l a t i o n s House f l y p o p u l a t i o n s were r e d u c e d up t o 94% >ird 89% i n t h e s m a l l and l a r g e s c a l e t e s t s , r e s p e c t i v e l y . The d a t a a l s o i n d i c a t e d t h a t l a r v a l d e n s i t i e s of the a s s o c i a t e d b e n e f i c i a l s o l d i e r f l y , Hermetia i l l u c e n s , were not r e d u c e d . T h i s i s an i m p o r t a n t observation s i n c e the most d e s i r a b l e c o n t r o l a g e n t s a r e t h o s e t h a t do not n e g a t i v e l y a f f e c t b e n e f i c i a l i n s e c t s w h i l e simultaneously c o n t r o l l i n g pest populations. Further a n a l y s i s o f t h e manure d u r i n g t h i s s t u d y i n d i c a t e d t h a t e r y t h r o s i n Β r a p i d l y d e g r a d e d under the e n c o u n t e r e d environmental conditions. This feature a l l e v i a t e s c o n c e r n s a b o u t p e s t i c i d e r e s i d u e s i n c h i c k e n manure w h i c h i s o f t e n u s e d as fertilizer. F o l l o w i n g t h i s s t u d y , e r y t h r o s i n Β was registered by S t e r l i n g Drug I n c . as I n t e r c e p t t o be m a r k e t e d as a l a r v i c i d e f o r house f l y c o n t r o l i n caged l a y e r facilities. L a t e r , t h i s compound was r e r e g i s t e r e d as S y n e r i d by H i l t o n D a v i s . A f t e r EPA r e g i s t r a t i o n o f the dye, H i l t o n D a v i s C h e m i c a l Company c o n d u c t e d f u r t h e r f i e l d t e s t s o f the p r o d u c t f o r l a r v a l f l y c o n t r o l i n C a l i f o r n i a , South C a r o l i n a , I n d i a n a and F l o r i d a . Results of these s t u d i e s , w i t h one e x c e p t i o n , r e m a i n u n p u b l i s h e d . The C a l i f o r n i a s t u d y e v a l u a t e d S y n e r i d and Synerid 100 (70% e r y t h r o s i n Β and 30% sodium f l u o r e s c e i n ) i n b o t h s m a l l and l a r g e p l o t s ( 2 7 - 2 8 ) . In s m a l l field t e s t s , t h r e e r a t e s o f S y n e r i d and S y n e r i d 100 were tested. O n l y one r a t e o f e a c h p r o d u c t was u s e d i n t h e large scale f i e l d tests. In b o t h t e s t s , t h e materials were a p p l i e d on a w e e k l y b a s i s . S y n e r i d 100 t r e a t m e n t s r e s u l t e d i n s i g n i f i c a n t l y fewer l a r v a e and a d u l t s r e l a t i v e t o the c o n t r o l s . Adult f l y p o p u l a t i o n s i n S y n e r i d t r e a t e d h o u s e s , however, d i d not d i f f e r s i g n i f i c a n t l y from e i t h e r S y n e r i d 100 or t h e control. The l a r g e s c a l e t e s t a l s o i n d i c a t e d t h a t

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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159

S y n e r i d 100 s i g n i f i c a n t l y r e d u c e d house f l y l a r v a l d e n s i t i e s r e l a t i v e t o c o n t r o l samples f r o m a n o t h e r house. D e s p i t e these s i g n i f i c a n t r e d u c t i o n s the l e v e l of f l y c o n t r o l was c o m m e r c i a l l y u n s a t i s f a c t o r y . A second study conducted i n C a l i f o r n i a , a l s o , showed t h a t w e e k l y s p r a y i n g o f t h e manure w i t h l a b e l l e d r a t e s o f S y n e r i d d i d n o t c o n t r o l house f l y l a r v a e ( 2 9 ) . A d u l t house f l y p o p u l a t i o n s , m o n i t o r e d w i t h s t i c k y t a p e s , were n e v e r below p r e t r e a t m e n t l e v e l s i n b o t h t h e c o n t r o l and S y n e r i d t r e a t e d h o u s e s . S i m i l a r r e s u l t s were o b t a i n e d i n l a r g e s c a l e f i e l d t e s t s c o n d u c t e d i n S o u t h C a r o l i n a ( N o l a n , I I I , M. P., Clemson U n i v e r s i t y , p e r s o n a l c o m m u n i c a t i o n ) and i n a s m a l l f i e l d t e s t a t t h e USDA-ARS l a b o r a t o r y i n Gainesvilie, Florida. The p r e l i m i n a r y f i e l d t e s t i n F l o r i d a was c o n d u c t e d in four outdoor f l y - p r o o i n 1985. E a c h roo c a g e s (.2 X .45 X .41 m) e a c h h o l d i n g two W h i t e L e g h o r n layers. Manure was a l l o w e d t o a c c u m u l a t e under t h e c a g e s and w i l d a d u l t house f l i e s were a l l o w e d a c c e s s t o the rooms t h r o u g h t h e d o o r s d u r i n g t h e c a r e o f t h e birds. Weekly t r e a t m e n t s o f e r y t h r o s i n Β were i n i t i a t e d and one o f f o u r t r e a t m e n t s was randomly a s s i g n e d ( u s i n g a random numbers t a b l e ) t o e a c h room. T r e a t m e n t s were as f o l l o w s : 139.5 mgr 209.3 mgr 294.0 mg/ 62.00 ml w a t e r / m , and no t r e a t m e n t - c o n t r o l . P o p u l a t i o n a s s e s s m e n t s were c o n d u c t e d by c o u n t i n g t h e number o f a d u l t house f l i e s c a u g h t on one s t i c k y t a p e i n a 24 hour period. The t a p e was hung under a randomly c h o s e n cage i n e a c h room. O n l y one t a p e was used p e r room t o i n s u r e t h a t t h e house f l y p o p u l a t i o n was n o t e l i m i n a t e d t h r o u g h i t s c a p t u r e on t h e t a p e . T a b l e I I i n d i c a t e s t h a t t h e t o t a l number o f a d u l t f l i e s c a u g h t by t h e s t i c k y t a p e i n e a c h room d i d n o t d e c r e a s e from p r e t r e a t m e n t l e v e l s a f t e r t h e a p p l i c a t i o n of e r t h y r o s i n B. S i n c e o n l y one t a p e was u s e d i n e a c h room and t h e e x p e r i m e n t was n o t r e p l i c a t e d t h e r e s u l t s were i n c o n c l u s i v e . However, c o m m u n i c a t i o n w i t h researchers conducting similar studies in C a l i f o r n i a , S o u t h C a r o l i n a , and I n d i a n a s t r o n g l y s u g g e s t e d t h a t t h e s e r e s u l t s were t y p i c a l . T h e r e f o r e , i t seemed p o i n t l e s s t o r e p l i c a t e such a l a b o r i n t e n s i v e study u n t i l more was known a b o u t 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 the p r o d u c t . The s l i g h t d e c l i n e i n p o p u l a t i o n l e v e l s t h a t was s e e n a t t h e end o f t h e s t u d y was most l i k e l y t h e r e s u l t o f p a r a s i t i s m and p r é d a t i o n by b e n e f i c i a l a r t h r o p o d s . N i n e t y p e r c e n t o f t h e pupae r e t u r n e d t o t h e l a b o r a t o r y t o m o n i t o r f o r f l y emergence were p a r a s i t i z e d by M u s c i d i f u r a x r a p t o r , a common house f l y p a r a s i t e . T h i s o b s e r v a t i o n c o r r o b o r a t e s t h e e a r l i e r s t u d y (26) w h i c h f o u n d e r y t h r o s i n Β had l i t t l e t o no e f f e c t on b e n e f i c i a l a r t h r o p o d s when t h e m a t e r i a l was s p r a y e d on t h e manure. 2

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D u r i n g t h i s s t u d y , however, a d u l t house f l i e s w i t h r e d abdomens were f o u n d r e s t i n g on t h e w a l l s o f t h e room f o l l o w i n g t h e s p r a y i n g o f manure w i t h e r y t h r o s i n B. We assumed t h a t t h e s e f l i e s were p r o d u c e d f r o m t r e a t e d l a r v a e , w h i c h a p p e a r e d p i n k i n c o l o r , and, would therefore ultimately die. However, none o f t h e s e f l i e s brought i n t o the l a b o r a t o r y d i e d . I n s t e a d t h e abdomens of a l l the f l i e s r e t u r n e d to t h e i r normal c o l o r w i t h i n 24 h; t h e f l i e s c o n t i n u e d t o l i v e and r e d f e c a l and o r a l s p o t s were o b s e r v e d i n t h e c a g e s . This indicated that f l i e s were a b l e t o s u c c e s s f u l l y c l e a n t h e i r abdomens o f the d y e .

Table

Julian Date 102 109 116 123 130 137 144 152 a

II.

T o t a l Number o f House F l y A d u l t s T r a p p e d on a S t i c k y Tape F o l l o w i n g t h e S p r a y i n g o f t h e Manur Β in a ( G a i n e s v i l l e , F l o r i d a 1985) 3

Treatment Medium Low 16 55 575 650 500 450 225 450 200 85 175 200 225 175 175 110

Control 161 600 275 550 150 400 250 175

High 37 830 650 500 300 225 200 150

T r e a t m e n t s were a p p l i e d on J u l i a n d a t e 102, 1985. R a t e s were: low=139.5 mg, medium=209.3 mg, and h i g h = 279.0 mg/62.00 ml w a t e r / m . 2

I t was l a t e r n o t e d t h a t p r i o r t o s p r a y i n g t h e manure no red-abdomened f l i e s were o b s e r v e d . However, w i t h i n 4 h o f s p r a y i n g t h e manure, a p p r o x i m a t e l y 60% o f t h e a d u l t h o u s e f l i e s had r e d abdomens. From t h e s e observations i t was c o n c l u d e d t h a t : 1) r e d f l i e s were n o t t h e r e s u l t o f t h e i r f e e d i n g on e r y t h r o s i n Β d u r i n g t h e i r l a r v a l s t a g e , 2) a d u l t house f l i e s would f e e d on t h e e r y t h r o s i n Β s p r a y w h i l e i t was wet, and 3) some o f t h e a d u l t s t h a t d i d i n g e s t t h e dye were a b l e t o e l i m i n a t e i t t h r o u g h d e f e c a t i o n and r e g u r g i t a t i o n . Adulticide Tests. The o b s e r v a t i o n s made i n t h e F l o r i d a f i e l d t e s t i n d i c a t e d t h a t S y n e r i d may i n f a c t be an e f f e c t i v e a d u l t i c i d e when f e d t o a d u l t f l i e s i n l a r g e enough amounts. T h e r e f o r e , e r y t h r o s i n Β was r e t u r n e d t o the l a b o r a t o r y f o r a c l o s e r e v a l u a t i o n o f i t s adulticidal activity. Research from these l a b o r a t o r y

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Photooxidative Dyes as insecticides

161

s t u d i e s i n d i c a t e d t h a t e r y t h r o s i n Β ( S y n e r i d ) showed more p r o m i s e as an a d u l t i c i d e t h a n a l a r v i c i d e (£) . F o l l o w i n g the l a b o r a t o r y s t u d i e s , s m a l l s c a l e f i e l d e v a l u a t i o n s o f e r y t h r o s i n Β as a l i q u i d a d u l t b a i t were c o n d u c t e d i n 1985 and 1986. The p u r p o s e o f the f i e l d t e s t i n g was t w o - f o l d : 1) t o e v a l u a t e c o n t r o l o f a d u l t house f l i e s w i t h e r y t h r o s i n Β and 2) t o o b s e r v e r a t e s o f f l y r e d u c t i o n when m u s c a l u r e was used i n c o n j u n c t i o n w i t h the dye. Laboratory experiments i n d i c a t e d that c e r t a i n l e t h a l c o n c e n t r a t i o n s of e r y t h r o s i n Β i n h i b i t e d i n g e s t i o n by house f l i e s . T h e r e f o r e , i t seemed n e c e s s a r y t o keep t h e f l i e s a t the b a i t s t a t i o n so t h a t a d u l t f l i e s would i n g e s t enough m a t e r i a l t o e n s u r e death. M u s c a l u r e was u s e d s i n c e i t a c t s as a f e e d i n g a r r e s t a n t f o r house f l i e s ( 3 0 - 3 1 ) . The r e s u l t s of t h e s e s t u d i e s have n o t y e t been p u b l i s h e d , t h e r e f o r e , t h e m a t e r i a l s and method Three of the fou t h e p r e v i o u s l y d e s c r i b e d s m a l l s c a l e f i e l d t e s t were u t i l i z e d i n t h i s experiment. C h i c k w a t e r i n g t o w e r s were u s e d as t h e b a i t s t a t i o n s . E a c h d e v i c e had a c i r c u l a r t r o u g h c o n t a i n i n g dye s o l u t i o n s . S t e r i l e c o t t o n was p r o v i d e d as a s u p p o r t medium so t h a t f l i e s c o u l d r e s t on i t and i n g e s t the f l u i d s . A l l b a i t s t a t i o n s were p l a c e d on t h e g r o u n d i n t h e s o u t h w e s t c o r n e r o f e a c h room c o n t a i n i n g the c h i c k e n c a g e s . S o l u t i o n s i n the w a t e r i n g t o w e r s were r e p l a c e d w e e k l y . One of t h r e e t r e a t m e n t s ( S y n e r i d , S y n e r i d + m u s c a l u r e and a c o n t r o l ) was p l a c e d i n e a c h room. A d u l t house f l y p o p u l a t i o n s i n a l l rooms were sampled d a i l y f o r f i v e d a y s p r e c e d i n g t r e a t m e n t w i t h a m o d i f i e d Scudder g r i d . From t h e day t h e t r e a t m e n t s were a p p l i e d , s a m p l i n g was done d a i l y f o r 3 weeks p o s t t r e a t m e n t e x c e p t on weekends. The s a m p l i n g p r o c e d u r e u s e d a m o d i f i e d 44 cm s q u a r e S c u d d e r g r i d c o n s i s t i n g o f 12 s t r i p s o f wood 44 cm l o n g and 2 cm wide s p a c e d 2 cm apart. The g r i d was r a n d o m l y p l a c e d on the g r o u n d i n e a c h room f o r 30 s e c o n d s and the number of f l i e s w h i c h r e s t e d on t h e g r i d a f t e r the 30 s e c o n d i n t e r v a l were counted. G r i d c o u n t s were r e p l i c a t e d 5 t i m e s i n e a c h room. R e s u l t s i n d i c a t e d t h a t a p p l i c a t i o n o f S y n e r i d (1% by volume e r y t h r o s i n Β b a i t ) w i t h m u s c a l u r e r e s u l t e d i n an a v e r a g e 95% and 51% r e d u c t i o n of house f l i e s from pretreatment l e v e l s i n s t u d i e s 1 and 2, r e s p e c t i v e l y ( T a b l e I I I and I V ) . Synerid without muscalure r e s u l t e d i n an a v e r a g e 67% r e d u c t i o n o f t h e i n i t i a l house f l y p o p u l a t i o n i n s t u d y 1 and 34% i n s t u d y 2. In c o n t r o l rooms house f l y p o p u l a t i o n s i n c r e a s e d from pretreatment l e v e l s by an a v e r a g e 10% i n s t u d y 1 and 31% i n s t u d y 2. A t p r e s e n t t h i s b r i n g s t h e f i e l d r e s e a r c h on e r y t h r o s i n Β f o r house f l y c o n t r o l u p - t o - d a t e . B e s i d e s the f i e l d e v a l u a t i o n s of e r y t h r o s i n Β f o r f l y c o n t r o l , a l i t t l e f i e l d work has been c o n d u c t e d a t e x a m i n i n g t h e

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

162

LIGHT-ACTIVATED PESTICIDES

feasibility species.

of u s i n g t h e s e dyes to c o n t r o l

Table

P e r c e n t R e d u c t i o n o f House F l y P o p u l a t i o n s F o l l o w i n g A p p l i c a t i o n of Synerid i n a Small S c a l e P o u l t r y F a c i l i t y ( F l o r i d a , 1985).

III.

insect

3

Day posttreatment 2 4 8 10 14 18 22 Average 3

other

Control -35.36 19.51 -28.04 -50.00 52.43 15.85

Percent Reduction Synerid+muscalure Synerid 79.45 60.58 95.89 56.20 87.67 71.53 95.89 64.96 91.78 34.03 91.78 80.29

When p e r c e n t r e d u c t i o n i s p r e c e d e d by a minus s i g n t h i s r e p r e s e n t s an i n c r e a s e by t h a t p e r c e n t i n t h e p o p u l a t i o n from t h e p r e t r e a t m e n t level.

Table

IV.

P e r c e n t R e d u c t i o n o f House F l y P o p u l a t i o n s F o l l o w i n g A p p l i c a t i o n of S y n e r i d i n a Small S c a l e P o u l t r y F a c i l i t y ( F l o r i d a , 1986).

Days 2 4 8 10 14 18 22 Average

54, .50 89..16 79..45 -16, .96 -235, .74 -10, .10 -58, .12 -31, .48

Reduction

15. 06 27. 38 53. 30 8. 02 -23. 28 54. 33 41. 09 34. 60

64. 70. 80. 78. 30. 58. 51. 51.

t h i s r e p r e s e n t s an i n c r e a s e by t h a t p e r c e n t p o p u l a t i o n from t h e p r e t r e a t m e n t level.

Mosquito F i e l d

3

Percent

60 28 50 08 66 72 88 26

i n the

Tests

Two s m a l l s c a l e f i e l d s t u d i e s have e v a l u a t e d t h e u s e o f f l u o r e s c e n t d y e s and e r y t h r o s i n Β f o r c o n t r o l o f m o s q u i t o l a r v a e . In t h e f i r s t s t u d y w h i c h was u n d e r t a k e n i n Germany, r e s u l t s i n d i c a t e d t h a t a c r i d i n e r e d and r o s e b e n g a l showed e f f i c a c y i n d i l u t i o n s o f 1:100,000 ( 3 2 ) . E x p o s u r e o f A n o p h e l e s l a r v a e t o a c r i d i n e r e d i n 6 o f 10 t r e a t e d s m a l l p o o l s r e s u l t e d i n 90 t o 100% m o r t a l i t y . In t h e o t h e r 4 p o o l s t h e r e s u l t s

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

10.

LEMKE ET AL.

Photooxidative Dyes as Insecticides

163

were v a r i a b l e (3J2) . The i n d i c a t i o n was t h a t t h e s e two d y e s may be s a f e and e f f e c t i v e m o s q u i t o c o n t r o l a g e n t s . R e c e n t l y C a r p e n t e r e t a l . (33) e v a l u a t e d e r y t h r o s i n Β f o r mosquito c o n t r o l . T h i s e x p e r i m e n t was c o n d u c t e d i n 30 1 χ 1 χ 0.05m h o l e s t h a t had been dug i n t h e ground. E a c h h o l e was l i n e d w i t h p l a s t i c s h e e t s and t h e n f i l l e d w i t h w e l l water f o r a 25 cm d e p t h . P r i o r to i n i t i a t i o n o f t h e e x p e r i m e n t 500 t o 600 f o u r t h i n s t a r Culex p i p i e n s quinquefasciatus l a r v a e were t r a n s f e r r e d i n t o each t e s t p l o t . L a r v a l samples were t a k e n u s i n g a d i p p e r 24 h a f t e r t h e a p p l i c a t i o n o f t h e e r y t h r o s i n B. The r e s u l t s showed t h a t a t 8.0 ppm 96% and 92% r e d u c t i o n s i n l a r v a l p o p u l a t i o n s c o u l d be s e e n i n s t u d i e s 2 and 3, r e s p e c t i v e l y . F i f t y percent c o n t r o l c o u l d be seen i n 24 h a t t r e a t m e n t r a t e s o f 0.5 t o 1.0 ppm ( 3 3 ) . The r e s u l t s o f s t u d y 1 i n d i c a t e d t h a t effective application c o n t r o l was d e p e n d e n treated. The pH o importan e f f e c t i v e c o n t r o l o f m o s q u i t o l a r v a e when u s i n g d y e s . Fire

Ant F i e l d

Experiments

Two s t u d i e s have a l s o been c o n d u c t e d t o a s s e s s t h e t o x i c i t y o f c e r t a i n d y e s on f i e l d c o l o n i e s o f i m p o r t e d f i r e ants. In t h e f i r s t s t u d y , f i e l d c o l l e c t e d mounds of S o l e n o p s i s r i c h t e r i , the black imported f i r e a n t , were dug up and b r o u g h t back t o t h e l a b o r a t o r y where t h e y were m a i n t a i n e d ( 3 £ ) . C o l o n i e s were t h e n f e d s o y b e a n o i l b a i t s w h i c h c o n t a i n e d t h e dye p h l o x i n B. I t was f o u n d t h a t p h l o x i n Β c a u s e d m o r t a l i t y t o c o l o n i e s and t h e amount o f t i m e i t took f o r d e a t h t o o c c u r was d e p e n d e n t on t h e amount o f l i g h t t o w h i c h t h e y were exposed. S i n c e t h e s e were f i e l d c o l l e c t e d c o l o n i e s , i t can be h y p o t h e s i z e d t h a t s i m i l a r r e s u l t s would be o b t a i n e d under a c t u a l f i e l d c o n d i t i o n s . However, i t would be n e c e s s a r y t o s e e i f t h i s p a r t i c u l a r b a i t i s as a t t r a c t i v e to f o r a g i n g ants. I f t h e queen does n o t i n g e s t t h e b a i t and d i e then c o l o n y l i f e w i l l go on undisturbed. T h e r e f o r e , i t i s e s s e n t i a l to study the r e a c t i o n s of f o r a g i n g f i r e ants i n the f i e l d to the m a t e r i a l , before extrapolating laboratory observations to f i e l d s i t u a t i o n s . A l a r g e s c a l e f i e l d t e s t was c o n d u c t e d by t h e a u t h o r i n o r d e r t o a s s e s s t h e e f f i c a c y o f a number o f b a i t s f o r c o n t r o l of i n d i v i d u a l red imported f i r e ant ( R I F A ) , S^ i n v i c t a , c o l o n i e s ( 3 5 ) . I n c l u d e d i n t h i s e v a l u a t i o n was a s o y b e a n o i l b a i t w h i c h c o n t a i n e d e r y t h r o s i n Β ( s u p p l i e d by t h e H i l t o n D a v i s Co., C i n c i n n a t i , OH). The c o n t r o l o f e r y t h r o s i n Β t r e a t e d c o l o n i e s was n o t s i g n i f i c a n t l y d i f f e r e n t (P>0.05) t h a n that of c o l o n i e s t r e a t e d with standard commercial b a i t Amdro ( T a b l e V) f o r 56 d a y s p o s t - t r e a t m e n t . The d e g r e e o f c o n t r o l w i t h a l l b a i t s , however, was n o t

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987. 71.2 46.4 59.6 15.7 14.0 53.3 0.0

21

a** a a b b a b

55.1 67.1 85.4 22.0 26.8 48.2 0.0

42

abc ab a ed cd be d 53.1 66.9 75.2 40.4 48.5 43.8 0.0

56

a a a a a a d

50.3 36.3 83.4 53.5 73.8 7.8 0.0

84

be c a be ab d d

44.3 38.1 78.3 53.3 73.8 8.7 0.0

112

c c a be ab d d

*Treatments were made 4 June 1984 ** Values expressed as percent of treated mounds that showed i n a c t i v i t y . Means within the same column, followed by the same l e t t e r , are not s i g n i f i cantly d i f f e r e n t (P> 0.05) according to the Least Squares Difference t e s t .

31 30 30 33 30 30 31

No. Mounds Treated

Days Post-Treatment*

F i e l d Evaluation of Baits for Use as Single Mound Treatments for Control of Red Imported F i r e Ants

Amdro Affirm A003 Affirm WO 02 Pro-Drone (31.Og) Pro-Drone (12.4g) Synerid Control

Bait

Table V.

10.

LEMKE ET AL.

165

Photooxidative Dyes as Insecticides

satisfactory. A d d i t i o n a l f i e l d t e s t s need c o n d u c t e d b e f o r e recommending t h i s p r o d u c t

t o be f o r RIFA.

Summary A l t h o u g h , t h e r e a r e many r e f e r e n c e s t o t h e b i o l o g i c a l a c t i v i t y o f p h o t o a c t i v e d y e s on i n s e c t s i n t h e l i t e r a t u r e , l i t t l e of i t addresses the e f f e c t i v e n e s s of them i n t h e f i e l d . I t i s i m p o r t a n t t o remember t h a t p o s i t i v e r e s u l t s i n t h e l a b o r a t o r y does n o t a s s u r e i t s success i n the f i e l d . Many e l e m e n t s such a s w e a t h e r , s u n l i g h t , h u m i d i t y , and pH c a n c a u s e p r o d u c t s t o be ineffective. The r e a l t e s t f o r t h e s e d y e s l i e s i n a d d i t i o n a l f i e l d t e s t s and i t i s hoped t h a t more f i e l d o r i e n t e d s t u d i e s w i l l be a t t e m p t e d . Commercial d e v e l o p m e n t has a l r e a d y shown t h a t such a p r o d u c t (Synerid) stands a r e l i a b l e informatio Laboratory developer together i n order t o assure the success of these products. Indeed t h e y a r e a most a t t r a c t i v e g r o u p o f i n s e c t i c i d e s when one c o n s i d e r s t h e s a f e t y and s e l e c t i v i t y o f t h e s e compounds. The d e v e l o p m e n t o f t h e s e p r o d u c t s i s j u s t i f i e d s i n c e t h e y a r e e x t r e m e l y s a f e w i t h many o f them b e i n g r e g i s t e r e d as f o o d a d d i t i v e s . Due t o t h i s s a f e t y , t h e r e i s low c o s t f o r t o x i c o l o g i c a l t e s t i n g i n o r d e r t o s a t i s f y EPA r e q u i r e m e n t s . Because l i t t l e t o x i c o l o g i c a l t e s t i n g i s needed, speedy r e g i s t r a t i o n o f t h e p r o d u c t by EPA c a n be a n t i c i p a t e d . In a d d i t i o n , few l a b e l r e s t r i c t i o n s a r e r e q u i r e d s i n c e there i s l i t t l e hazard to the a p p l i c a t o r , c r o p s , domestic a n i m a l s , w i l d l i f e or fish. I n an age o f h e a l t h and e n v i r o n m e n t a l l y c o n s c i e n c e i n d i v i d u a l s , t h e s e p r o d u c t s c a n be used without controversy. Literature 1. 2. 3. 4. 5. 6.

7.

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Yoho, T . P . ; B u t l e r , L.; Weaver, J. J . Econ. Entomol. 1971, 64, 972-3. Yoho, T . P . ; Weaver, J. E.; B u t l e r , L . E n v i r o n . Entomol. 1973, 2, 1092-6. Yoho, T . P . ; B u t l e r , L.; Weaver, J. E . E n v i r o n . Entomol. 1976, 5, 203-4. K o e h l e r , P. G.; P a t t e r s o n , R. S. J . Econ. Entomol. 1986, 79, 1023-26. Fondren, Jr., J. E.; H e i t z , J. R. E n v i r o n . Entomol. 1978, 7, 843-6. Broome, J. R . ; Callaham, M. F.; Lewis, L . Α.; Ladner, M. C.; H e i t z , J. R. Comp. Biochem. Physiol. 1975, 51, 117-21. Callaham, M. F.; Broom, J. R . ; L i n d i g , Ο. H.; H e i t z , J. R. E n v i r o n . Entomol. 1975, 4, 837-41.

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LIGHT-ACTIVATED PESTICIDES Broome, J. R . ; Callaham, M. F.; Poe, N. R . ; H e i t z , J. R. C h e m . - B i o l . I n t e r a c t . 1976, 14, 203-6. B a r h i e r i , A. R i v i s t a d i M a l a r i o l o g i c a 1928, 7, 456-63. Barbosa, P . ; P e t e r s , T. M. Mosq. News 1969, 29, 243-51. P i m p r i k a r , G. D . ; Norment, Jr., B. R . ; H e i t z , J. R. E n v i r o n . Entomol. 1979, 8, 856-9. F a i r b r o t h e r , T. Ε. Ph.D. T h e s i s , M i s s i s s i p p i State U n i v e r s i t y , S t a r k v i l l e , 1978. S a k u r a i , H.; H e i t z , J. R. E n v i r o n . Entomol. 1982, 11, 467-70. P i m p r i k a r , G. D . ; Noe, B. L.; Norment, B. R . ; H e i t z , J. R. J . Econ. Entomol. 1980, 73, 785-8. Graham, K . ; Wranger, E.; Sasan, L . H. Can. J. Zool. 1972, 50, 1625-9. L a v i a l l e , M . ; Dumortier B C R Acad Sc P a r i s 1978, 287, 875-8 Clement, S. L.; G . ; H e i t z , J. R. J . Econ. Entomol. 1980, 73, 390-392. L u , F . C.; L a v a l l e , A. Can. Pharm. J. 1964, 30, 530. Butterworth, K. R . ; Gaunt, I . F.; Grasso, P . ; G a n g o l l i , S . D . F d . Cosmet. T o x i c o l . 1976, 14, 525-31. RTECS, NIOSHA, Supt. of Documents, U . S . Government P r i n t O f f i c e : Washington, DC, 1983. Farm Chemicals Handbook, 1977, 5th ed. H e i t z , J. R. Disposal and Decontamination of Pesticides; Kennedy, M. V., American Chemical S o c i e t y : Washington, DC, 1978; No. 73, p 35-48. A x t e l l , R. C.; Rutz, D. A. Entomol Soc. Am. M i s c . Publ. 1986, 61, 88-100. H i n k l e , Ν. C.; Sheppard, D. C.; Nolan, Jr., M. P. J. Econ. Entomol. 1985, 78, 722-4. Bloomcamp, L . M.S. T h e s i s , U n i v e r s i t y of F l o r i d a , G a i n e s v i l l e , 1986. P i m p r i k a r , G. D., Fondren, Jr., J. E.; H e i t z , J. R. E n v i r o n . Entomol. 1980, 9, 53-8. Meyer, J. Α . ; Mullens, Β . Α . ; Rooney, W. F.; Rodriguez, J. L . J. A g r i c . Entomol. 1986, 2, 351-7. Meyer, J. Α . ; Mullens, Β . Α . ; Rooney, W. F. Progress in P o u l t r y ; Univ. C a l i f o r n i a Coop. Extension S e r v . , 1986, No. 31, 6 pp. Meyer, J. Α . ; Bradley, F . Progress in P o u l t r y ; Univ. C a l i f o r n i a Coop. Extension S e r v . , 1986, No. 34, 3 pp. C a r l s o n , D. Α . ; Mayer, M. S . ; S i l h a c e k , D. L.; James, J. D . ; Beroza, M . ; Bierl, B. A. Science 1971, 174, 76-8. C a r l s o n , D. Α . ; Beroza, M. E n v i r o n . Entomol. 1973, 2, 555-9.

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Schildmacher, H. B i o l . Z e n t r . 1950, 69, 468-77. Carpenter, T. L.; R e s p i c i o , Ν. C.; H e i t z , J. R. J. Econ. Entomol. 1985, 78, 232-7. David, R. M . ; H e i t z , J. R. J . A g r i c . Food Chem. 1978, 26, 99-101. Lemke, L . A. Ph.D T h e s i s , Clemson U n i v e r s i t y , Clemson, SC, 1986.

RECEIVED February 11, 1987

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

C h a p t e r 11

Photodecomposition of Naturally Occurring Biocides Y. Yoke Marchant ARCO Plant Cell Research Institute, 6560 Trinity Court, Dublin, CA 94568

Light-activated biocide occurring compound molecules. These chemicals are active against microorganisms, insects and nematodes, as well as s n a i l s and f i s h in v i t r o . Many are p o t e n t i a l l y useful as commercial pesticides, a property p a r t i c u l a r l y enhanced by evidence of rapid biodegradability in the environment. The mechanisms of photodegradation and the factors which influence t h i s process w i l l be discussed in t h i s review.

Many k i n d s o f compounds have been r e p o r t e d t o be t o x i c t o b i o l o g i c a l systems i n the presence o f l i g h t under a e r o b i c and a n a e r o b i c conditions. In photodynamic r e a c t i o n s the p h o t o n - e x c i t e d s e n s i t i z e r m o l e c u l e t r a n s f e r s i t s e x c i t a t i o n energy t o oxygen, g e n e r a t i n g t h e s i n g l e t s t a t e which may s u b s e q u e n t l y r e a c t w i t h p h o s p h o l i p i d s , p r o t e i n s and s t e r o l s o f c e l l u l a r membranes. A s t r u c t u r a l l y d i v e r s e group o f p h y t o c h e m i c a l s i s o l a t e d from p l a n t s has been r e p o r t e d t o e x h i b i t b i o c i d a l a c t i v i t y towards v i r u s e s , b a c t e r i a , f u n g i , nematodes and i n s e c t s i n the presence o f s u n l i g h t o r UV-A r a d i a t i o n (320-400nm) (±zD- Such compounds i n c l u d e v a r i o u s a l k a l o i d s ( 5 . 6 ) , acetophenones ( J ) , extended a n t h r a q u i n o n e s ( 4 ) , f u r a n o c o u m a r i n s furochromones ( 1 0 ) , s t r a i g h t - c h a i n and a r o m a t i c p o l y a c e t y l enes, and t h i o p h e n e s ( e . g . 11-15). In a d d i t i o n , s y n t h e t i c xanthene dyes such a s r o s e bengal a r e w e l l known p h o t o a c t i v e p e s t i c i d e s and f i s h p o i s o n s (16-20). R e p r e s e n t a t i v e examples o f these compounds a r e shown i n F i g u r e s 1 and 2. In the s e a r c h f o r e f f e c t i v e and e n v i r o n m e n t a l l y n o n t o x i c b i o l o g i c a l c o n t r o l a g e n t s , the b i o d e g r a d a b i l i t y o f a c t i v e compounds i s an e s s e n t i a l p r a c t i c a l c o n s i d e r a t i o n . A p e s t i c i d e o r f u n g i c i d e which has performed i t s f u n c t i o n s h o u l d s u b s e q u e n t l y decompose i n t o m o i e t i e s which have no l o n g term e f f e c t s on the environment. S i n c e t h i s i s a symposium on l i g h t - a c t i v a t e d p e s t i c i d e s , t h i s d i s c u s s i o n a d d r e s s e s the i s s u e s o f l i g h t s t a b i l i t y and p h o t o d e g r a d a t i o n o f n a t u r a l l y - o c c u r r i n g b i o c i d e s , p a r t i c u l a r l y p h o t o a c t i v e ones, w i t h

0097-6156/87/0339-0168$06.00/0 © 1987 American Chemical Society

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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F i g u r e 1. N a t u r a l l y o c c u r r i n g p h o t o a c t i v e compounds. I . Dictamnine (Dictamnus a l b a ) I I . H y p e r i c i n (Hypericum s p p . ) , I I I . 8-Methoxypsoralen (Rutaceae, A p i a c e a e ) , IV. K h e l l i n (Ammi s p p . ) , V. Harman, V I . 6-Methoxyeuparin ( E n c e l i a s p p . ) , V I I . A l p h a - t e r t h i e n y l (Tagetes s p p . ) V I I I . P h e n y l h e p t a d i y n e ene ( B i d e n s s p p . ) , IX. P h e n y l h e p t a t r i y n e ( B i d e n s s p p . ) , X. Heptadeca t e t r a e n e d i y n e ( B i d e n s s p p . ) . t

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In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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r e f e r e n c e t o t h e work on t h e p h o t o d e c o m p o s i t i o n o f t h e h a l o g e n a t e d xanthene dyes. Degradation o f Non-photoactive Natural

Pesticides

Many h e r b i c i d e s and p e s t i c i d e s i n t h e environment a r e degraded by UV r a d i a t i o n from the s u n . The exposure o f a z a d i r a c h t i n s o l u t i o n s t o s u n l i g h t caused a r a p i d d e c r e a s e i n a n t i f e e d i n g potency a g a i n s t f i r s t i n s t a r l a r v a e o f Spodoptera f r u g i p e r d a ( J . E. Smith) and r e s u l t e d i n complete d e s t r u c t i o n o f t h e compound and i t s a c t i v i t y a f t e r 16 days. HPLC a n a l y s e s o f t h e exposed s o l u t i o n s showed no t r a c e s o f a z a d i r a c h t i n ( 2 1 ) . The a d d i t i o n o f v a r i o u s p l a n t o i l s , such as neem and c a s t o r , t o t h e t e s t s o l u t i o n s a f f o r d e d some p r o t e c t i o n (<25J) a g a i n s t p h o t o d e g r a d a t i o n a l t h o u g h whether t h i s was due t o t h e e x c l u s i o n o f oxygen from the medium was n o t i n v e s t i g a t e d . When a p p l i e d t o soybean l e a v e s i n f i e l d t e s t s , crude e x t r a c t s o f neem p r e v e n t e d damag soybea foliag b Popilli b e e t l e s f o r about two weeks comparable t o c o n t r o l p l a n t s a t t r i b u t e d t o t h e p h o t o d e g r a d a t i o n and r e s u l t a n t l o s s o f a n t i f e e d a n t a c t i v i t y o f a z a d i r a c h t i n i n t h e crude e x t r a c t s ( 2 2 ) . In a study o f r o t e n o n e , t h e p r i n c i p a l i n s e c t i c i d a l component o f D e r r i s r o o t , Bowman e t a l . found t h a t f i f t y p e r c e n t o f t h e compound i n a l c o h o l i c s o l u t i o n was photodegraded i n l i g h t t o t h e d e m e t h y l a t e d and reduced d e r i v a t i v e s r o t e n o l o n e , dehydrorotenone and rotenonone. Only rotenone a t o r near t h e s u r f a c e o f t h e s o l u t i o n s was a v a i l a b l e f o r r e a c t i o n which s u g g e s t s t h a t compounds formed a t t h e s u r f a c e e i t h e r e x c l u d e oxygen and/or d i m i n i s h l i g h t a b s o r p t i o n ( 2 ^ ) . A m u l t i t u d e o f o t h e r rotenone p h o t o d e c o m p o s i t i o n p r o d u c t s w i t h p o s s i b l e c a r c i n o g e n i c p r o p e r t i e s have a l s o been observed and c h a r a c t e r i z e d ( 2 4 ) . In a d d i t i o n , some o f t h e components o f t h e p y r e t h r i n s found i n Chrysanthemum c i n e r a r i a e f o l i u m ( T r e v i r . ) V i s . a l s o degrade i n s u n l i g h t w i t h subsequent l o s s o f i n s e c t i c i d a l a c t i v i t y . Decomposition i n v o l v e s complex i s o m e r i z a t i o n / rearrangement r e a c t i o n s accompanied by e x t e n s i v e p o l y m e r i z a t i o n (25.26). Photoactive

Xanthene Dyes

The l i g h t s t a b i l i t y o f dyed t e x t i l e s and the p r o p e r t i e s o f dye m o l e c u l e s have been the s u b j e c t o f study f o r a number o f y e a r s and numerous r e p o r t s e x i s t on the p h o t o c h e m i c a l c h a r a c t e r i s t i c s o f dyes which may s e r v e as u s e f u l model systems f o r n a t u r a l p r o d u c t s ( 2 7 ) . The p h o t o s t a b i l i t y o f a l l UV a b s o r b i n g m o l e c u l e s depends on a number o f f a c t o r s . These i n c l u d e t h e c h e m i c a l s t r u c t u r e and p h o t o p h y s i c a l p r o p e r t i e s o f t h e compound, i t s c o n c e n t r a t i o n i n the medium, t h e n a t u r e o f t h i s medium, l i g h t q u a n t i t y and q u a l i t y , temperature and o t h e r e n v i r o n m e n t a l c o n d i t i o n s o f exposure, and t h e presence o f o t h e r r e a c t i v e s p e c i e s i n t h e medium and/or environment ( 2 8 ) . Although the p r e c i s e r o l e o f oxygen i n t h e f a d i n g o f f a b r i c dyes has been the s u b j e c t o f c o n t r o v e r s y , under normal exposure c o n d i t i o n s the presence o f oxygen a c c e l e r a t e s the r a t e o f p h o t o d e c o m p o s i t i o n o f dyes. I t i s now g e n e r a l l y a c c e p t e d t h a t t h e t r i p l e t s t a t e o f many dyes can c a t a l y z e the f o r m a t i o n o f s i n g l e t oxygen which l e a d s t o t h e

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p h o t o o x i d a t i o n o f the s u b s t r a t e s i n which they a r e d i s p e r s e d and even to the s e l f - s e n s i t i z e d p h o t o o x i d a t i v e r e a c t i o n s o f a z o n a p t h o l dyes i n low c o n c e n t r a t i o n s (27.29.30). S u b s t i t u t e d xanthene d y e s , p a r t i c u l a r l y the h a l o g e n a t e d f l u o r e s c e i n d e r i v a t i v e s , have been shown to be h i g h l y p h o t o a c t i v e t o i n s e c t s i n v i t r o (16-19). T o x i c i t y i s due to the a b i l i t y o f the dyes t o absorb l i g h t , form t r i p l e t e x c i t e d s t a t e s and s u b s e q u e n t l y t r a n s f e r t h i s energy t o form r e a c t i v e s i n g l e t oxygen. Activity i n c r e a s e s w i t h the phosphorescence o f the dye m o l e c u l e and w i t h the number and a t o m i c weight o f the s u b s t i t u e n t h a l o g e n s . These f a c t o r s i n c r e a s e the r e l a t i v e p o p u l a t i o n o f the f i r s t e x c i t e d t r i p l e t s t a t e o f the dye upon i l l u m i n a t i o n and enhance the s e n s i t i z a t i o n o f ground s t a t e oxygen to the s i n g l e t s t a t e (31.32) ( F i g u r e 2 ) . Tonogai e t a l . (20,33) s t u d i e d the t o x i c i t y o f f o u r xanthene dyes t o f i s h and found t h a t t o x i c i t y was g r e a t e r a f t e r i r r a d i a t i o n . The major p r o d u c t s o f a n a e r o b i c p h o t o d e c o m p o s i t i o n were f l u o r e s c e i n and t e t r a c h l o r o f l u o r e s c e i e r y t h r o s i n , rose bengal d a t a show t h a t i r r a d i a t i o n i n the absence o f oxygen causes d e h a l o g e n a t i o n w i t h o u t the breakdown o f the b a s i c xanthene s k e l e t o n and s u g g e s t s t h a t t o x i c i t y i s most l i k e l y caused by the l i b e r a t e d halogens. Xanthene dyes i n aqueous oxygenated s o l u t i o n s photodegrade when exposed to v i s i b l e l i g h t and the r a t e o f d e g r a d a t i o n depends on oxygen c o n c e n t r a t i o n . Photobleaching f o l l o w s f i r s t order k i n e t i c s o n l y when dye c o n c e n t r a t i o n i s low r e l a t i v e t o oxygen c o n c e n t r a t i o n (21). The v i s i b l e a b s o r p t i o n spectrum o f r o s e bengal d i s a p p e a r s c o m p l e t e l y as p h o t o d e c o m p o s i t i o n o c c u r s and a complex m i x t u r e o f i n t e r m e d i a t e s and p r o d u c t s r e s u l t s . Photodegraded r o s e b e n g a l does not k i l l h o u s e f l i e s (Musca d o m e s t i c a ) or i n h i b i t the growth o f S t a p h y l o c o c c u s aureus Rosen, and B a c i l l u s cereus Fr.& F r . ( 3 2 ) . A c c o r d i n g to H e i t z and W i l s o n (22) who t e s t e d the s u s c e p t i b i l i t y o f a s e r i e s o f dyes to p h o t o d e g r a d a t i o n , the two dyes which c o n t a i n no h a l o g e n , rhodamine B and f l u o r e s c e i n ( X I V ) , were most r e s i s t a n t t o p h o t o d e g r a d a t i o n . Iodine and/or bromine atoms on the upper r i n g system f a c i l i t a t e the r e a c t i o n w h i l e c h l o r i n e atoms on the lower r i n g r e t a r d the p h o t o d e g r a d a t i o n r e a c t i o n . In a d d i t i o n , s u s c e p t i b i l i t y to p h o t o d e g r a d a t i o n i s p o s i t i v e l y c o r r e l a t e d w i t h phosphorescence o f the dye. M o l e c u l e s which phosphoresce can assume the t r i p l e t s t a t e more r e a d i l y and hence c a t a l y z e the g e n e r a t i o n o f s i n g l e t oxygen. I t i s c l e a r t h a t s i n g l e t oxygen i s r e s p o n s i b l e f o r photoa c t i v i t y as w e l l as f o r the p h o t o d e c o m p o s i t i o n o f the photodynamic xanthene dyes. N e v e r t h e l e s s , many o t h e r dyes are s u s c e p t i b l e t o p h o t o b l e a c h i n g i n the absence o f oxygen, p a r t i c u l a r l y i n the presence o f e l e c t r o n donors and i n s o l u t i o n s c o n t a i n i n g s p e c i e s w i t h a r e a d i l y e x t r a c t a b l e hydrogen atom. The mechanism o f p h o t o r e d u c t i o n o f t e n i n v o l v e s the f o r m a t i o n o f r a d i c a l or semiquinone i n t e r m e d i a t e s d e t e c t a b l e as s t r o n g t r a n s i e n t s and, i n the case o f azo dyes, may l e a d t o d e s t r u c t i o n o f the chromophore (28,29.34).

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Thiophenes

Many p o l y a c e t y l e n e s , n o t a b l y p h e n y l h e p t a t r i y n e ( I X ) and i t s b i o s y n t h e t i c d e r i v a t i v e a l p h a - t e r t h i e n y l (VII) are a l s o t o x i c to b i o l o g i c a l systems i n the presence o f UV-A r a d i a t i o n (320-400nm) ( 1 . 2 ) . U n l i k e the l i n e a r furanocoumarins whose e f f e c t s can be e x p l a i n e d by the photoinduced m o d i f i c a t i o n o f DNA (8), the l i p o p h i l i c n a t u r e o f V I I and IX suggests t h a t they may p a r t i t i o n i n t o membrane b i l a y e r s and thus e x e r t t h e i r e f f e c t s p r i m a r i l y on c e l l membranes. A l p h a - t e r t h i e n y l a c t s as a t y p i c a l Type I I photodynamic s e n s i t i z e r , r e q u i r i n g oxygen f o r i t s a c t i v i t y , w h i l e photos e n s i t i z a t i o n by p h e n y l h e p t a t r i y n e o c c u r s under both a e r o b i c and a n a e r o b i c c o n d i t i o n s (11.35-37). They have been shown t o i n a c t i v a t e membrane-bound enzymes and cause i n c r e a s e d p e r m e a b i l i t y t o K+ i o n s and subsequent h e m o l y s i s i n e r y t h r o c y t e s upon i r r a d i a t i o n (35.3840). Both a l p h a - t e r t h i e n y l and p h e n y l h e p t a t r i y n e enhance the permeability of multilamella e v i d e n t l y by two d i f f e r e n c h e m i c a l and b i o p h y s i c a a n a l y s e system expose to UV-A i n d i c a t e t h a t a l p h a - t e r t h i e n y l a f f e c t s membrane p e r m e a b i l i t y by a l t e r i n g a c y l s i d e c h a i n s i n the hydrocarbon r e g i o n s i n eggphosphatidylcholine liposomes. Photopolymerization of phenyl h e p t a t r i y n e i s the p o s t u l a t e d cause o f enhanced p e r m e a b i l i t y i n d i s t e a r o y l p h o s p h a t i d y l c h o l i n e liposomes ( 4 2 ) . In a d d i t i o n t o b e i n g t h e r m a l l y s e n s i t i v e , a c e t y l e n e s a r e a l s o known t o be u n s t a b l e i n l i g h t and i n aqueous s o l u t i o n s ( 1.43-45). In a study on the p h o t o t o x i c i t y o f s e l e c t e d p o l y a c e t y l e n e s t o the p h y l l o p l a n e y e a s t C r y p t o c o c c u s l a u r e n t i i ( K u f f . ) S k i n n e r , aqueous s o l u t i o n s o f up to 10 ug/mL o f t e s t compounds were used t o p r e p a r e dose response c u r v e s i n an a e r o b i c m i c r o t i t e r assay ( 4 6 ) . Ultra v i o l e t i r r a d i a t i o n o f l o n g e r than f i v e minutes d u r a t i o n seemed t o cause breakdown o f phenyldiyne-ene ( V I I I ) and the C17 s t r a i g h t c h a i n compound ( X ) , w i t h a concommitant i n c r e a s e i n the p e r c e n t s u r v i v a l o f C. l a u r e n t i i . A l p h a - t e r t h i e n y l ( V I I ) and p h e n y l h e p t a t r i y n e ( I X ) were not degraded a f t e r 20 minutes o f r a d i a t i o n under those t e s t conditions. In v i t r o and iri v i v o d e g r a d a t i o n e x p e r i m e n t s by McLachlan e t a l . (36) showed t h a t s t r a i g h t c h a i n a c e t y l e n e s decomposed most r a p i d l y f o l l o w e d by a r o m a t i c a c e t y l e n e s , and l a s t l y , t h i o p h e n e s . Photodecomposition was marked by a c o l l a p s e o f the UV s p e c t r a o f V I I and IX and by the r a p i d f o r m a t i o n and d i s a p p e a r a n c e o f i n t e r m e d i a t e s p e c i e s i n o t h e r compounds. Although the p h o t o d e g r a d a t i o n o f s t r a i g h t c h a i n and a r o m a t i c a c e t y l e n e s does not r e q u i r e oxygen, thiophene d e c o m p o s i t i o n i s a e r o b i c . The a u t h o r s suggest t h a t t h e r e i s g r e a t e r e f f i c i e n c y i n the t r a n s f e r o f i m p i n g i n g l i g h t energy t o s i n g l e t oxygen i n t h i o p h e n e s and t h a t a nonphotodynamic p r o c e s s competes s u c c e s s f u l l y w i t h s i n g l e t oxygen g e n e r a t i o n i n the o t h e r p o l y a c e t y l e n e s r e s u l t i n g i n a h i g h e r number o f m o l e c u l a r rearrangement e v e n t s . Conclusions Numerous d e t a i l s a r e known about n a t u r a l l y - o c c u r r i n g p h o t o s e n s i t i z e r s and t h e i r potent i n v i t r o e f f e c t s on b i o l o g i c a l

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systems. These p r o p e r t i e s have been e x p l o i t e d i n some c a s e s , f o r example, 8-methoxypsoralen ( I I I ) has been used t o t r e a t p s o r i a s i s , v i t i l i g o and o t h e r s k i n d i s o r d e r s f o r a number o f y e a r s (42), and r e c e n t l y , a p a t e n t a p p l i c a t i o n f o r t h e p o s s i b l e commercial use o f a l p h a - t e r t h i e n y l a s a mosquito and b l a c k f l y l a r v i c i d e was f i l e d (48). P o l y a c e t y l e n e s and t h i o p h e n e s a r e t o x i c t o numerous o r g a n i s m s a t low c o n c e n t r a t i o n s and t h e i r p o t e n t i a l a s commercial i n s e c t i c i d e s , f u n g i c i d e s , p i s c i c i d e s and m o l l u s c i d e s i s f u r t h e r enhanced by e v i d e n c e o f r a p i d b i o d e g r a d a b i l i t y o f most compounds i n aqueous s o l u t i o n and s u n l i g h t . The c h e m i c a l n a t u r e and l i f e t i m e s o f p h o t o d e c o m p o s i t i o n i n t e r m e d i a t e s and p r o d u c t s has n o t been t h o r o u g h l y e x p l o r e d b u t c l e a r l y t h i s must be a d d r e s s e d b e f o r e t h e s e compounds can be used i n t h e f i e l d . Literature Cited 1. 2. 3. 4 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Towers, G. H. N. Ca 1984, 62, 2900-11. Towers, G. H. N. J Downum, K. R.; Rodriguez 1986, 12, 823-34. Knox, J . P.; Dodge, A. D. Phytochemistry. 1985, 24, 889-96. Towers, G. H. N.; Abramovski, Z. J . Nat. Prod. 1983, 46, 57681. Ashwood-Smith, M. J . ; Towers, G. H. N.; Abramovski, Z. ; Poulton, G. A.; L i u , M. Mutat. Res. 1982, 102, 401-12. Proksch, P.; Proksch, M.; Towers, G. H. N.; Rodriguez, E. J. Nat. Prod. 1983, 46, 331-34. Song, P.-S.; Tapley, K.J. J r . Photochem. Photobiol. 1979, 29, 1177-97 Berenbaum, M. Science 1978. 201, 532-34. Abeysekera, B. F.; Abramovski, Z.; Towers, G. H. N. Photochem. Photobiol. 1983, 38, 311-15. Arnason, J . T.; Wat, C. K.; Downum, K. R.; Yamamoto, E.; Graham, E.; Towers, G. H. N. Can. J . Microbiol. 1980, 26, 698705. Champagne, D. E.; Arnason, J . T., Philogene, B. J . R.; Morand, P.; Lam, J . J . Chem. Ecol. 1986, 12, 835-58. Hudson J . B.; Graham, E. A.; Towers, G. H. N. Photochem. Photobiol. 1982, 36, 181-86. Marchant, Y. Y.; Towers, G. H. N. Biochem. Syst. Ecol. 1986a, in press. Downum, K. R. In Natural Plant Resistance to Pests: Roles of Allelochemicals; Green, M.; Hedin, P. A., Eds.; American Chemical Society, Washington, DC, 1986; pp 197-205. Carpenter, T. L.; Respicio, N. C.; Heitz, J . R. Environ. Entomol. 1984, 13, 1366-70. Carpenter, T. L.; Mundie, T. G.; Ross, J . H.; Heitz, J . R. Environ. Entomol. 1981, 10, 953-56. Pimprikar, G. D.; Fondren, J . E. J r . ; Heitz, J . R. Environ. Entomol. 1980, 9, 53-8. Heitz, J . R. In Insecticide Mode of Action; Coats, J . R., Ed.; Academic Press, New York, 1982; pp 429-57. Tonogai, Y.; Ito, Y.; Iwaida, M.; T a t i , M.; Ose, Y.; Sato, T. J. Toxicol. S c i . 1979, 4, 115-26.

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Stokes, J . B.; Redfern, R. E. J . Environ. S c i . Health 1982, A l l , 57-65. 22. Ladd, T. L. J r . ; Jacobson, M.; B u r i f f , C. R. J . Econ. Entomol. 1978, 71, 810-14. 23. Bowman, M. C.; Holder, C. L.; Bone, L.I. J . Assoc. Off. Anal. Chem. 1978, 61, 1445-55. 24. Cheng, H. M.; Yamamoto, I.; Casida, J . E. J . Agric. Food Chem. 1972, 20, 850-6. 25. Bullivant, M. J . ; Pattenden, G. Pyrethrum Post 1973, 13, 64-75. 26. Bullivant, M. J . ; Pattenden, G. Pyrethrum Post 1971, 11, 72-9. 27. S i n c l a i r , R. Y. Photochem. Photobiol. 1980, 31, 627-9. 28. Evans, N. A.; Stapleton, I. W. In Chemistry of Synthetic Dyes. Venkataraman, K. Ed.; Academic Press, New York, 1978, 8, 221-77. 29. McKellar, J . F. Radiat. Res. Rev. 1971, 3, 141-65. 30. Bentley, P.; McKellar, J . F. Rev. Prog. Coloration 1974, 5,3348. 31. Wilson, W. W.; Heitz 17. 32. Heitz, J . R.; Wilson, W. W. In Disposal and Decontamination of Pesticides. Kennedy, M. V., Ed.; American Chemical Society, Washington, DC, 1978, pp 35-48. 33. Tonogai, Y.; Iwaida, M.; T a t i , M.; Ose, Y.; Sato, T. J. Toxicol. S c i . 1978, 3, 205-14. 34. Kellman, A.;. Lion, Y.; Photochem. Photobiol. 1979. 29, 217-22. 35. Wat, C. K.; MacRae, W. D.; Yamamoto, E.; Towers, G. H. N.; Lam. J . Photochem. Photobiol. 1980, 32, 167-72. 36. McLachlan, D.; Arnason, J . T.; Lam, J . Photochem. Photobiol. 1984, 39, 117-82. 37. Weir, D.; Scaiano, J . C. ; Arnason, J . T.; Evans, C. Photochem. Photobiol. 1985, 42, 223-30. 38. Yamamoto, E.; Wat, C. K.; MacRae, W. D.; Towers, G. H. N. FEBS Lett. 1979, 107, 134-6. 39. Bakker, J . ; Gommers, F. J . ; Nieuwenhuis, I.; Wynberg, H. J. B i o l . Chem. 1979, 254, 1841-4. 40. MacRae, W. D.; Irwin, D. A. J . Bisalputra, T.; Towers, G. H. N. Photobiochem. Photobiophys. 1980, 1, 309-18. 41. McRae, D. G.; Yamamoto, E.; Towers, G. H. N. Biochim. Biophys. Acta 1985, 821, 488-96. 42. McRae, D. G.; Webb, M.; Marchant, Y. Y.; Towers, G. H. N.; Thompson, J . E. Biochim. Biophys. Acta 1986, submitted. 43. Anchel, M.; Polatnick, J . ; Kavanagh, F. Arch. Biochem. 1950, 25, 208-20. 44. Celmer, W. D.; Solomons, I. A. Amer. Chem. Soc. J . 1953, 751, 372-76. 45. Bohlmann, F.; Burkhardt, T.; Zdero, C. Naturally Occurring Acetylenes. Academic Press, London, 1973. 46. Marchant, Y. Y.; Towers, G. H. N. Biochem. Syst. Ecol. 1986b. in press 47. Warin, A. P., Carruthers, J . A. C l i n . Exptl. Dermatol. 1976, 1, 181-189. 48. Towers,. G. H. N.; Arnason, J . T.; Wat, C. K.; Lambert, J . D. H. Can. Pat. 1984, 1, 173,743. RECEIVED

November 20, 1986

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 12

α-Terthienyl

as a P h o t o a c t i v e Insecticide:

T o x i c E f f e c t s on N o n t a r g e t

Organisms

Jacques Kagan, William J. Bennett, Edgard D. Kagan, Jacqueline L. Maas, Susan A. Sweeney, Isabelle A. Kagan, Emmanuelle Seigneurie, and Vitautas Bindokas Department of Chemistry, University of Illinois, Chicago, IL 60680

Alpha-terthienyl Laboratory experiment produce light-dependent toxic effects i n non-target organisms. The results obtained with the fish Pimephales promelas (fathead minnow), tadpoles of Rana pipiens and Hyla c r u c i f e r , and water f l e a s (Daphnia magna) are reviewed. They cast a doubt upon the published claim that alpha-terthienyl had an a c t i v i t y against non -target organisms low enough to allow its use f o r mosquito control in the f i e l d . Greater selectivity was encountered with other phototoxic molecules (Figure 1). Although t h e i d e a l p e s t i c i d e i s expected t o d i s p l a y p e r f e c t s e l e c t i v i t y against i t s intended target organism, few commercially available pesticides do unfortunately. T o x i c i t y against non-target organisms, i n c l u d i n g humans, must t h e r e f o r e be determined under r e a l i s t i c c o n d i t i o n s i n o r d e r t o d e c i d e whether the p o t e n t i a l benefits i n the use of a new product outweigh the r i s k s . Light-Dependent i n s e c t i c i d e s have had l i m i t e d commercial use t o date, and l i t t l e i s known about t h e i r s e l e c t i v i t y . Alpha-terthienyl, 1, i s a m o l e c u l e which has d i s p l a y e d t o x i c i t y i n a v a r i e t y o f organisms, such as b a c t e r i a , v i r u s e s , f u n g i , nematodes, human erythrocytes and human skin, eggs and larvae of insects, algae and plants (1). However, i t was reported a t a recent symposium that, i n f i e l d a p p l i c a t i o n s , a l p h a - t e r t h i e n y l used i n mosquito control had very low a c t i v i t y against non-target organisms (2). Because we had a l r e a d y d e s c r i b e d the l i g h t - d e p e n d e n t t o x i c i t y of the compound i n Rana pipiens tadpoles {3), and because f o r a number o f years we had routinely used aquatic organisms f o r monitoring the a c t i v i t y of new p h o t o t o x i c m o l e c u l e s , we thought t h a t a c r i t i c a l survey o f t h e p h o t o t o x i c i t y o f a l p h a - t e r t h i e n y l i n n o n - t a r g e t organisms was desirable. I n t h i s r e v i e w we comment on t h e l i g h t - d e p e n d e n t t o x i c i t y of 1 i n the mosquito Aedes aegypti, and compare i t t o that i n other environmentally relevant aquatic organisms, ttie laboratory f

0097-6156/87/0339-0176$06.00/0 © 1987 American Chemical Society

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

KAGAN ET AL.

a-Terthienyl as a Photoactive Insecticide

F i g u r e 1. Structure of the phototoxic campcamds mentioned.

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data were obtained by placing the organisms i n t o water containing the desired amount of s e n s i t i z e r , waiting up t o 1 h and placing the v e s s e l under a bank o f 8 tubes (RPR-3500A from the Southern New England U l t r a v i o l e t Co, Hamden, CT) emitting a t 320-400 nm, with a maximum a t 350 nm. The tubes were p l a c e d h o r i z o n t a l l y 7 t o 9 cm above t t e water, and the l i g h t i n t e n s i t y a t t h i s distance was about 13 W m . The i r r a d i a t i o n time ranged from 30 min t o 1 h. While our experiments were not i n t e n d e d t o r e p l a c e a c t u a l exposure o f the organisms t o s u n l i g h t under e n v i r o n m e n t a l l y relevant conditions, they were meant t o p r o v i d e a r a n k i n g o f the s e n s i t i v i t y o f the d i f f e r e n t organisms exposed t o UV l i g h t under s i m i l a r , reproducible, c o n d i t i o n s . The r e s u l t s s h o u l d be q u a l i t a t i v e l y s i m i l a r t o those obtained with sunlight. f

The P h o t o i n s e c t i c i d a l A c t i v i t y of Alpha-terthienyl We used the mosquito Aede a c t i v i t y of alpha-terthieny from those r e p o r t e d i n e a r l i e r s t u d i e s (4, 15). I n o r d e r t o d e t e r mine acute t o x i c i t y data and t o f o l l o w the development of organisms which had been t r e a t e d a t s p e c i f i c s t a g e s , we used a procedure r e c e n t l y t e s t e d w i t h o t h e r p h o t o s e n s i t i z e r s (J5). Egg sheets were placed i n water i n the niorning and kept i n an incubator a t 28 ° C In the afternoon f i r s t i n s t a r larvae were collected, and were incubated overnight a t room temperature i n the presence of 1. A f t e r 30 min of i r r a d i a t i o n w i t h UV l i g h t (320-400 nm), the s u r v i v i n g l a r v a e were p l a c e d i n p e t r i d i s h e s w i t h a d d i t i o n a l water, f e d , and observed d a i l y u n t i l adults emerged i n dark controls. From the shape of the s u r v i v a l curves a t d i f f e r e n t i n i t i a l concentrations, we intended t o determine the e x t e n t and t i m i n g o f any delayed t o x i c i t y . However, p r a c t i c a l l y a l l the l a r v a e which s u r v i v e d 24 h reached adulthood (2)F i g u r e 2 shows the s u r v i v a l p r o f i l e s a t 0.005 mg/L, the highest ccncentraticn a t which s u r v i v a l was observed, and a t 0.0009 mg/L compared t o 0.03 mg/L i n the dark (very s i m i l a r t o the p r o f i l e o b t a i n e d w i t h u n t r e a t e d l a r v a e ) . Except f o r the amplitudes, the curves a r e v e r y s i m i l a r i n shape. The absence o f d e l a y e d e f f e c t s suggest t h a t the mechanism o f p h o t o t o x i c i t y does not i n v o l v e a d r a s t i c modification of nucleic acids, which would have been l i k e l y t o i m p a i r m o l t i n g and/or emergence t o a d u l t s . The c o n c l u s i o n t h a t the light-^dependent t o x i c i t y of alpha-terthienyl r e s u l t s from damage to membranes rather than t o nucleic acids i s supported experimen t a l l y by the recent r e s u l t s of Tuvescn e t a l . with bacteria (8). The absence of delayed t o x i c i t y which we observed with alpha-terthienyl a l s o c o n t r a s t s w i t h the d e l a y e d e f f e c t s r e g i s t e r e d w i t h f u r a n o coumarins i n s i m i l a r experiments (6). These compounds are known t o produce photoadducts with DNA. F o u r t h i n s t a r l a r v a e , not s u r p r i s i n g l y , were much more r e s i s t a n t t o the photosensitized treatment than f i r s t i n s t a r larvae. Our L C v a l u e s were 0.002 and 0.45 mg/L w i t h 1 s t and 4th i n s t a r larvae respectively, i n general accord with the r e s u l t s of Arnason e t a l (4) and Wat e t a l . (J5). A comparison o f the p h o t o t o x i c l e v e l s of 1 i n A. a e g y p t i and A. i n t r u d e n s under i d e n t i c a l c o n d i t i o n s has not been made with f i r s t - i n s tar larvae, but there seems t o be q u a l i t a t i v e agreement f o r older larvae of these two species (9). 5 0

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

12.

a-Terthienyl as a Photoactive Insecticide

KAGAN ET AL.

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DAYS F i g u r e 2. S u r v i v i n g l a r v a e ( s o l i d l i n e s ) , pupae (broken l i n e s ) and a d u l t s ( d o t t e d l i n e s ) o b t a i n e d f r o m f i r s t - i n s t a r l a r v a e o r A. a e g y p t i i n t h e dark ( c l o s e d c i r c l e s ) and i r r a d i a t e d f o r 30 min. (open c i r c l e s ) , a s a f u n c t i o n o f t i m e . The l a r v a e were i n c u b a t e d o v e r n i g h t w i t h 1 p r i o r t o t h e i r i r r a d i a t i o n . The c o n c e n t r a t i o n s o f 1, w h i c h were 0.03 ( t o p ) , 0.005 ( m i d d l e ) , and 0.0009 mg/L (bottom), a r e shewn on a l o g a r i t h m i c s c a l e . (Repro duced w i t h p e r m i s s i o n f r e m R e f e r e n c e 7. C o p y r i g h t 1987 Plenum.)

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Since older larvae were more r e s i s t a n t t o the light-dependent e f f e c t o f a l p h a - t e r t h i e n y l than younger ones, we expected t h a t t r e a t i n g s t i l l younger larvae would produce more dramatic r e s u l t s . T h i s e x p e c t a t i o n was not f u l f i l l e d . F o r example, l a r v a e about 2-h o l d were t r e a t e d as above w i t h a l p h a - t e r t h i e n y l , except t h a t the incubation time was shortened from about 14 h t o 30 min, while the i r r a d i a t i o n time was kept a t 30 min. In these experiments the larvae were much younger when i r r a d i a t e d , but t h e i r exposure t o the s e n s i t i z e r was c o r r e s p o n d i n g l y s h o r t e r . The L C Q v a l u e s observed 24 h l a t e r were 0.075 mg/L i n the dark c o n t r o l s , and 0.0016 i n the exposed larvae. Measurement the p h o t o t o x i c i t y o f 1 toward mosquito eggs was more d e l i c a t e because t h i s compound has an appreciable t o x i c i t y t o larvae i n the dark, an e f f e c t l i k e l y t o be a t i t s maximum with newly hatched larvae. By performing inicroscopic examinations of the eggs, we established that 1 was not phototoxic a t concentrations up t o 6.7 mg/L. A t t h i s c o n c e n t r a t i o n were k i l l e d by the s e n s i t i z e r note that the lack of photoovicida a c t i v i t y aegypt s not a g e n e r a l c h a r a c t e r i s t i c o f 1, s i n c e a v e r y h i g h p h o t o o v i c i d a l a c t i v i t y has been documented i n Drosophila melanoqaster (10). Unexpected r e s u l t s came from the study of the pupae, believed t o be immune t o photodamage by 1 (4h Our i n i t i a l r e s u l t s indeed agreed with t h i s conclusion since a l l the pupae were s t i l l a l i v e 24 h a f t e r phototreatment with 6.7 mg/L of 1, but highly irreproducible r e s u l t s were o b t a i n e d when the i r r a d i a t e d pupae were observed through adult emergence. F i n a l l y , we recognized that the age of the pupae a t the time of treatment was an important variable, and deter mined the s u r v i v a l curves shown i n F i g u r e 3. A t 1 o r 2 days o f age, the pupae were quite s e n s i t i v e t o the phototreatment with 1 (LC^Q = 0.06 mg/L)r but three-day-old pupae were no longer affected. To our knowledge, t h i s photoinduced e f f e c t o f 1 i s the f i r s t example o f i n s e c t pupae k i l l e d with a photoactive i n s e c t i c i d e . Since pupae are usually quite r e s i s t a n t t o pesticides, t h i s a c t i v i t y makes 1 an even more i n t e r e s t i n g photopesticide than o r i g i n a l l y suspected. 5

The Photoactivity of Alpha-Terthienyl i n Tadpoles Several years ago, we observed that 1 was phototoxic i n the immature frog Rana pipiens (3). The experiments were performed with up t o 2 h of sunlight, which i s highly v a r i a b l e i n our area, and the s u r v i v a l was recorded immediately a f t e r i r r a d i a t i o n . Even with such l i m i t e d exposure, 1 showed s i g n i f i c a n t t o x i c i t y . The L C C Q v a l u e f o r acute phototoxicity was 0.065 mg/L a f t e r 30 min, and 0.1)25 mg/L a f t e r 2 h of i r r a d i a t i o n . Wishing t o o b t a i n more complete d a t a , we r e c e n t l y c o l l e c t e d t a d p o l e s comparable i n development t o those o f R^ p i p i e n s used e a r l i e r . They t u r n e d o u t t o be t a d p o l e s o f H y l a c r u c i f e r , and they were used f o r measuring the phototoxicity of 1 i n sunlight and i n UV l i g h t . In these experiments, the i n i t i a l incubation time was 1 h and the i r r a d i a t i o n time was a l s o 1 h, but the s u r v i v a l was determined 24 h a f t e r treatment. Again, a l t h o u g h t h e s e a r e s h o r t d u r a t i o n s compared t o those l i k e l y t o be f a c e d by organisms i n nature, the L C Q values were very impressive, about 0.003 mg/L i n both s e r i e s of 5

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experiments (Figure 4). This phototoxicity l e v e l of 1 i s comparable t o the 24-h s u r v i v a l v a l u e (0.002 mg/L) observed w i t h the f i r s t i n s t a r l a r v a e o f A. a e g y p t i (2). The a n a l y s i s o f the p h o t o t o x i c i t y o f 1 i n immature mosquitos from d i f f e r e n t genera and/or d i f f e r e n t s p e c i e s under c a r e f u l l y c o n t r o l l e d c o n d i t i o n s has n o t y e t been r e p o r t e d . I t was found (Borovsky, L i n l e y , and Kagan, u n p u b l i s h e d r e s u l t s ) t h a t , i n the presence o f s u n l i g h t , the d i f f e r e n c e i n p h o t o t o x i c response i n larvae of A^_ aegypti, A. taeniorhynchus, and Culex quinquefasciatus was q u i t e l a r g e , w i t h A^ a e g y p t i b e i n g the most a f f e c t e d a t the l o w e s t c o n c e n t r a t i o n s . A r e c e n t r e p o r t gave a 24-h L C Q v a l u e o f 0.0275 mg/L when A. a t r o p a l p u s was exposed t o 1 and UV l i g h t (11). Because of differences i n conditions ( l i g h t i n t e n s i t y , duration of the exposures, age of the larvae), d i r e c t comparison with our work i s meaningless. 5

Phototoxicity of Alpha-Terthieny We investigated the t o x i c i t y of 1 i n f i s h using the fathead minnow (Pimephales promelas) (12), which i s commonly a v a i l a b l e and has been used e x t e n s i v e l y i n t o x i c o l o g y . Again, our e x p e r i m e n t a l conditions d i d not approximate f i e l d conditions, which would have p r o v i d e d much l o n g e r exposures. No t o x i c i t y was observed a t c o n c e n t r a t i o n s as h i g h as 10 mg/L when the f i s h were kept i n the dark. However, w i t h 30 min o f i n c u b a t i o n f o l l o w e d by 30 min o f i r r a d i a t i o n , the 24-h L C Q v a l u e was found t o be about 0.05 mg/L when UV l i g h t was used, ana about 0.02 mg/L when sunlight was used. In order t o c a l i b r a t e the magnitude of t h i s e f f e c t , we measured on our f i s h the l i g h t - i n d e p e n d e n t t o x i c i t y o f rotenone, one o f the best-known f i s h p o i s o n s , and found i t s 24-h L C g v a l u e t o be 0.04 mg/L. In other words, 1 i s a f i s h poison a t least twice as potent as rotenone. We were i n t e r e s t e d i n d e t e r m i n i n g how the exposure t i m e t o 1 b e f o r e i r r a d i a t i o n a f f e c t e d the s u r v i v a l o f the f i s h . Longer i n c u b a t i o n s may be expected t o i n c r e a s e the amount o f s e n s i t i z e r p i c k e d up by the f i s h (thereby i n c r e a s i n g the t o x i c i t y ) , but they a l s o allow more time f o r depuration (thereby decreasing the t o x i c i t y of the s e n s i t i z e r ) . The actual s u r v i v a l p r o f i l e showed a ininimum a t 2 h (Figure 5), as would be expected i f the depuration process were s l o w e r than the uptake, t a k i n g i n t o account the l i m i t e d amount o f s e n s i t i z e r a v a i l a b l e t o the f i s h {1). The term depuration has been used here t o i m p l y the c l e a r i n g o f the t o x i n from the organisms' system, w i t h o u t r e g a r d f o r the a c t u a l mechanisms, which c o u l d involve, f o r example, d e t o x i f i c a t i o n and/or excretion. Further work on the mechanism o f t h i s d e p u r a t i o n p r o c e s s w i l l be h i g h l y desirable. The mechanism f o r the phototoxicity of 1 i n f i s h i s not known. However, we established that d i r e c t contact was important. In these experiments (I), water f l e a s (Daphnia magna) were exposed t o known doses o f 1 p r i o r t o b e i n g f e d t o f i s h which, i n t u r n , were exposed t o UV l i g h t . N e g l i g i b l e p h o t o t o x i c i t y was observed i n the t r e a t e d fish. 5

5

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Concentration (ppm) F i g u r e 3. S u r v i v a l o f A. a e g y p t i pupae t h r o u g h a d u l t emergence, f o l l o w i n g a 30-min. i n c u b a t i o n and 30-min. i r r a d i a t i o n i n t h e p r e s e n c e o f 1. The age o f t h e pupae a t t h e t i m e o f i r r a d i a t i o n was 0-1 (open c i r c l e s ) , 1-2 (open s q u a r e s ) , and 2-3 day ( c l o s e d circles). (Reproduced w i t h p e r m i s s i o n from R e f e r e n c e 7. Copy r i g h t 1987 Plenum.)

0001

001

01

I

CONCENTRATION (mg/U F i g u r e 4. Survival (24 h) o f immature Hyla c r u c i f e r as a function of the concentration o f 1 (on a logarithmic scale). The incubation time was 1 h, and the i r r a d i a t i o n 1 h, with sunlight (circles) o r with UV (squares).

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Phototoxicity of 1 i n Daphnia magna The a c t i v i t y of 1 was a l s o measured i n the water f l e a D, magna, a standard organism i n t c x i c o l o g i c a l studies. In the dark, no t o x i c i t y was n o t i c e d a t c o n c e n t r a t i o n s below 7 mg/L and, i n the c o n t r o l experiments, the UV l i g h t alone had no e f f e c t en the s u r v i v a l of the organisms. In the light-dependent s t u d i e s , the organisms were incubated f o r 1 h with 1, and then i r r a d i a t e d with UV l i g h t f o r 1 h. The s u r v i v a l was recorded 24 h l a t e r . The L C value i n these conditions was 0.0013 mg/L (Figure 6) (12). 5 0

Mechanistic

considerations

The d e t a i l e d b i o l o g i c a l mechanism f o r the p h o t o t o x i c i t y o f 1 i n a q u a t i c organisms i s c o m p l e t e l y unknown. In another example of phototoxicity i n f i s h , that of anthracene, extensive damage t o the s k i n and g i l l s were r e p o r t e appeared t o generate d e t a i l e d analysis remain i s reasonable t o expect that 1, an excellent s i n g l e t oxygen s e n s i t i z e r (14), damages c e l l components through oxidative processes. The r e s u l t s obtained with A^_ aegypti suggest that n u c l e i c acids are not affected, since very l i t t l e delayed m o r t a l i t y was observed. In view of other examples where c e l l membranes were damaged by 1, such as i n human e r y t h r o c y t e s (15), l i p i d damage may be expected. Recent r e s u l t s from Tuveson et a l . provide additional confirmation, since the same k i n e t i c s of p h o t o i n a c t i v a t i o n w i t h 1 were found i n f o u r mutant s t r a i n s of K_ c o l i which contained a l l four possible combina t i o n s of genes c o n t r o l l i n g e x c i s i o n p r o f i c i e n c y and s e n s i t i v i t y to oxidative damage (8). We a l s o attempted t o p r o t e c t organisms from the p h o t o t o x i c e f f e c t s of 1 by using aqueous solutions containing beta-carotene, a standard s i n g l e t oxygen quencher known t o r e a c t a t d i f f u s i o n c o n t r o l l e d r a t e s (16j. L i t t l e o r no p r o t e c t i o n c o u l d be demonstrated i n e i t h e r mosquito l a r v a e o r Daphnia, u n l a s s a l a r g e excess of carotene was used. For example, fourth i n s t a r larvae of A. a e g y p t i were incubated f o r 30 min i n a 0.19 mg/L s o l u t i o n o f 1 i n the presence of v a r y i n g amounts of beta-carotene. F i g u r e 7 shows the 24-h s u r v i v a l curve, i n d i c a t i n g t h a t about 30 mg/L o f b e t a carotene produces 50% protection, about an 80-fold molar excess of b e t a - c a r o t e n e over 1. S i m i l a r s t u d i e s w i t h Daphnia i n 5 ug/L of 1 showed that 50% protection was produced by 50 mg/L of beta-carotene, nearly a 5000-fold molar excess (Figure 8). In these two examples, i t i s l i k e l y that standard s i n g l e t oxygen quenching was not a promi nent r e a c t i o n ; perhaps the p r o t e c t i n g e f f e c t of beta-carotene was s i m p l y due t o f i l t e r i n g o f the UV l i g h t , an a p p l i c a t i o n o f Beer's law. I t i s u s e f u l t o note i n t h i s r e g a r d t h a t s i n g l e t oxygen quenching experiments have usually been much less successful when performed i n v i v o than i n v i t r o . In an extreme case, some s i n g l e t oxygen quenchers added t o the d i e t were observed t o enhance even the phototoxic e f f e c t of xanthene dyes i n f l i e s , although a very modest protection with beta-carotene was noted (17).

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F i g u r e 5. Percent survival of P^ promelas as a function of incubation time. In a l l cases the concentration of 1 was 0.1 mg/Lf and the i r r a d i a t i o n time 30 min. The survival was recorded 24 h a f t e r the i r r a d i a t i o n s . (Reproduced with permission from Ref 12. Copyright 1987 Pergamcn)

CONCENTRATION (mg/L)

F i g u r e 6. Survival (24 h) o f a_ magna, with 1 h incubation and 1 h i r r a d i a t i o n . The concentration scale f o r 1 i s l o g a r i t h m i c (Reproduced with permission from Ref 12. Copyright 1987 Pergamon)

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to

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'oo

CONCENTRATION (mg/L)

F i g u r e 7. Survival o incubated with 1 (0.02 mg/L) f o r 30 min and i r r a d i a t e d f o r 30 min i n the presence of varying concentrations o f beta-carotene (shown on a logarithmic scale), 1 h (broken line) and 24 h ( s o l i d line) a f t e r i r r a d i a t i o n . (Reproduced with permission from Ref 12. Gopyright 1987 Pergamon)

n

no

CONCENTRATION (mg/L)

F i g u r e 8. Survival o f magna incubated f o r 1 h i n the presence of 1.2 mg/L of 1, and i r r a d i a t e d f o r 1 h. The experiments were conducted i n the presence o f varying concentrations o f betacarotene (shown on a logarithmic scale), and the r e s u l t s were recorded immediately a f t e r i r r a d i a t i o n ( s o l i d line) and 24 h l a t e r (broken l i n e ) . The dotted l i n e represents the s u r v i v a l i n the dark, i n the absence o f 1. (Reproduced with permission from Ref 12. Copyright 1987 Pergamon)

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Alpha-terthienyl As A Phototoxic Insecticide; Is I t Selective? The r e s u l t s o f our l a b o r a t o r y experiments suggest t h a t 1 i s not a s e l e c t i v e photoinsecticide that i t has very s i g n i f i c a n t a c t i v i t y i n aquatic organisms, and that i t i s premature t o advocate i t s use f o r mosquito control. Opposite views have been expressed (2, 18). Two points a t issue i n t h a t work concern the i n t e r p r e t a t i o n of the v e r y h i g h photo t o x i c i t y i n Daphnia (higher than i n mosquito larvae) and the lack of p h o t o t o x i c i t y o f 1 i n t r o u t , i n r e l a t i o n t o the s u i t a b i l i t y of t h e c h e m i c a l f o r f i e l d use. The s t a t e m e n t (L8) t h a t "under l a b o r a t o r y c o n d i t i o n s Daphnia .... s u r v i v e d 1 and UV t r e a t m e n t w i t h o u t any s i g n i f i c a n t o r v i s i b l e s i g n s o f i n t o x i c a t i o n " i s supported neither by the o r i g i n a l r e s u l t s (18) nor by ours. Further studies may be required before passing a f i n a l judgment on the s u i t a b i l i t y o f 1 f o r i n s e c t c o n t r o l . I t i s c o n c e i v a b l e t h a t trout have some protectio depuratio mechanis availabl fathead ininnows. However l i k e l y t o be a s s o c i a t e exposing the f i s h t o the c h e m i c a l . Instead o f adding an ethanol s o l u t i o n t o the water c o n t a i n i n g the f i s h (18), we used d i m e t h y l s u l f o x i d e as c a r r i e r f o r the v e r y hydrophobic 1, and we mixed the water thoroughly before adding the f i s h . The compound p r e c i p i t a t e s out o f s o l u t i o n a l m o s t i m m e d i a t e l y i n the f o r m e r procedure, but apparently not i n the l a t t e r . In the absence of mechanical mixing, the s e n s i t i z e r which p r e c i p i t a t e s forms a f i l m on the water, and any f i s h remaining away from the surface can be immune from phototoxic e f f e c t s d u r i n g s h o r t - t e r m experiments. In s m a l l v e s s e l s under l a b o r a t o r y c o n d i t i o n s , we c o u l d not demonstrate any s i g n i f i c a n t difference i n the outcome whether EtOH or DMSO was used. Even with a modest s c a l e - u p o f the h o l d i n g v e s s e l (from 400 t o 1000 mL), the f i s h f r e q u e n t l y swam by the s u r f a c e , and a l l d i s p l a y e d equal s u s c e p t i b i l i t y t o the l i g h t treatment. In l a r g e r tanks o r under f i e l d c o n d i t i o n s , however, p a r t i c u l a r l y i n the absence o f wind o r currents, the r e s u l t s perhaps could be quite d i f f e r e n t . F i n a l l y , the k i n e t i c s of the various processes a t play cannot be ignored. To a f i s h r e m a i n i n g a t a f i x e d p o s i t i o n i n the water, the r i s k s of exposure are time-dependent, during the period when the chemical p r e c i p i t a t i n g a t the surface establishes equilibrium with the b u l k o f the water ( i n such experiments, the r a t i o o f s u r f a c e area t o volume o f the s o l u t i o n should be q u i t e important). Irradiated before the s e n s i t i z e r had a chance of reaching i t , a f i s h w i l l s u r v i v e . Introduced d i r e c t l y i n t o an homogeneous s o l u t i o n of the s e n s i t i z e r , t h i s f i s h w i l l be a t much greater r i s k much sooner. The d i f f e r e n c e w i l l a l s o depend on the t i m e span between the i n t r o d u c t i o n o f the c h e m i c a l and the b e g i n n i n g o f the experiment, which i n c l u d e s the a c t u a l c o n t a c t t i m e o f the f i s h w i t h the chemical. In our experiments we placed 5 f i s h i n an homogeneous mixture o f 1 i n 0.4 L o f water and i r r a d i a t e d 0.5 h l a t e r . In the work w i t h t r o u t 1 was added t o the s u r f a c e o f ca. 40 L o f water c o n t a i n i n g 10 f i s h which were i r r a d i a t e d 1 h l a t e r (18). The lack of phototoxicity f o r 1 reported i n trout could be simply the r e s u l t of i n s u f f i c i e n t c o n t a c t between the c h e m i c a l and the organisms, r a t h e r than t o b i o l o g i c a l immunity. r

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I t i s i n t e r e s t i n g t o note t h a t w h i l e t h e p r e c i p i t a t i o n o f 1 which o c c u r s when an e t h a n o l s o l u t i o n i s added t o water s h o u l d decrease t h e p h o t o t o x i c e f f e c t s i n f i s h and most o t h e r a q u a t i c organisms, i t should a c t u a l l y enhance the phototoxicity i n mosquito l a r v a e . S i n c e these must come t o the s u r f a c e o f the water i n o r d e r t o breathe, they a r e exposed t h e r e t o d i s p r o p o r t i o n a t e l y h i g h concentrations o f the hydrophobic chemical. Under n a t u r a l c o n d i t i o n s , c o n s i d e r a t i o n must be g i v e n t o situations not e a s i l y controlled i n laboratory o r f i e l d t r i a l s . For example, phototoxic damage t o organisms obviously depends upon t h e i r exposure t o UV l i g h t . In waters whose absorption c o e f f i c i e n t follows a steep v e r t i c a l gradient, the organisms tested would gain protec t i o n by r e m a i n i n g near the bottom d u r i n g daytime. They c o u l d a l s o gain some protection by remaining i n shaded areas o r under shelters. F i n a l l y , some components o f n a t u r a l waters c o u l d perhaps p r o v i d e protection from photodynamic damage by a c t i n g as quenchers, a f f e c t i n g e i t h e r the l i f e t i m f sensitizer' excited state that f s i n g l e t oxygen o r other t o x i c agents. The r a t e o f d e p u r a t i o n i s another f a c t o r i m p o r t a n t t o t h e s u r v i v a l of an aquatic organism i n contact with a photosensitizer. Since l i t t l e i s known i n t h i s area, we placed fathead minnows i n a solution containing 0.05 mg/L of 1. A f t e r 30 min, they were divided i n t o two groups. One group was i r r a d i a t e d f o r 30 min, and 50% o f these f i s h were dead 24 h l a t e r . The o t h e r group was t r a n s f e r r e d i n t o clean water and kept there f o r various lengths of time before exposure t o UV l i g h t . As shown i n F i g u r e 9, t h e p h o t o s e n s i t i z i n g e f f e c t o f 1 were o f f r a p i d l y : the f i s h were no longer a t r i s k a f t e r 3 h of depuration. This observation should give optimism t o f i s h which become a c c i d e n t a l l y exposed t o a p h o t o a c t i v e p e s t i c i d e . By heading toward u n p o l l u t e d water, p a r t i c u l a r l y under cover o f darkness, they stand a good chance of f u l l recovery. Likewise, other a q u a t i c organisms may w e l l have r a p i d d e p u r a t i o n mechanisms. However, a l l may n o t be e q u a l l y a b l e t o escape r a p i d l y from a contaminated area and t o avoid exposure t o l i g h t before completing the d e p u r a t i o n process. F o r example mosquito l a r v a e , which must f r e q u e n t l y come t o t h e s u r f a c e i n o r d e r t o breathe, a r e v e r y u n l i k e l y t o t r a v e l as f a s t as f i s h away from contaminated s u r f a c e water toward deeper and c l e a n e r areas. Here a g a i n , t h e dynamic aspects of the photosensitization under non-equilibrium conditions a r e h i g h l y important, and they p r o v i d e some t a r g e t s e l e c t i v i t y i n the use of l i g h t - a c t i v a t e d pesticides as mosquito l a r v i c i d e s . Conclusions To our knowledge, there i s not y e t one molecule whose phototoxicity has been proved t o be r e s t r i c t e d t o one s i n g l e target organism. The statement t h a t "1 i s a h i g h l y e f f e c t i v e l a r v i c i d e w i t h a c c e p t a b l e nontarget e f f e c t s " (18) needs further objective evaluation. I t would be p a r t i c u l a r l y important t o obtain a f u l l set of data f o r target as w e l l as f o r non-target organisms under f i e l d conditions. In o u r r e c e n t s t u d i e s , we encountered two o t h e r p h o t o t o x i c molecules which, under i d e n t i c a l experimental conditions, produced

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F i g u r e 9. S u r v i v a l (24 h) o f P, promelas exposed t o 1 (0.1 mg/L) f o r 30 min, transferred i n t o clean water, and i r r a d i a t e d f o r 30 min a f t e r the delay shown.

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e x a c t l y the same L C C Q v a l u e as 1 i n f i r s t - i n s t a r l a r v a e o f A. aegypti. They are 5-(4-chlorcphenyl)-2 3--diphenylthiophene ( 2 ) and benzo[a]pyrene ( 3 ) . The former (UBI-T930, M i c r o m i t e ) has an extremely low t o x i c i t y i n mammalian organisms, and was developed by Uniroyal Co. as an a c a r i c i d e (19-20). The l a t t e r , on the other hand, i s a well-known c a r c i n o g e n . I n some r e c e n t work we demonstrated that, contrary to e a r l i e r opinions, p h o t o t o x i c i t y i n p o l y c y c l i c a r o m a t i c hydrocarbons (PAH's) i s not n e c e s s a r i l y a s s o c i a t e d w i t h t h e i r c a r c i n o g e n i c i t y , and t h a t t h e environmental impact o f p o l l u t i o n with PAH's associated with t h e i r light-dependent a c t i v i t y remains t o be assessed (21-23). Comparing the phototoxicity of 1, 2, and 3, we have uncovered a degree of s e l e c t i v i t y which suggests that further research designed t o a m p l i f y t h i s p r o p e r t y c o u l d be p r o f i t a b l e . I t i s p a r t i c u l a r l y s t r i k i n g w i t h r e s p e c t t o the f i s h P^ promelas. While 1, i n the presence o f UV, ranks among the v e r y b e s t f i s h p o i s o n s known, we could not demonstrate an conditions (this represent t h r e e o r d e r s o f magnitude). F i g u r e 10 a l s o i l l u s t r a t e s another example of d i f f e r e n t i a l phototoxicity, observed with tadpoles of H. c r u c i f e r . There i s a difference of roughly one order of magnitude i n the L C Q v a l u e s f o r 24-h s u r v i v a l i n going from 1 ( L C Q = 0.02 mg/L), t o 3 ( L C - 0.4 mg/L), and t o 2 ( L C = 3 mg/L). Future research on the design of photoactive i n s e c t i c i d e s w i l l c e r t a i n l y lead t o more a c t i v e compounds possessing greater s e l e c t i v i t y . In such studies, the discovery of molecules without a c t i v i t y i n one important organism, d e s p i t e f a v o r a b l e s p e c t r a l p r o p e r t i e s , may turn out t o be of greater value than the f i n d i n g of yet one more phototoxic compound. f

5

5

5 0

5 0

F i g u r e 10. Survival (24 h) of tadpoles of IL_ c r u c i f e r exposed t o 1 ( s o l i d c i r c l e s ) , 2 (open c i r c l e s ) , and 3 (squares) f o r 1 h and i r r a d i a t e d f o r 1 h. The concentration scale i s logarithmic.

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Acknowledgments We are grateful t o Profs G. B. Craig, Jr., University of Notre Dame, D. Bardack, H. E. Buhse, J r . , and R. L. W i l l e y , U n i v e r s i t y o f I l l i n o i s a t Chicago, f o r assistance with the organisms used i n t h i s research. Our e a r l y work was supported by the National I n s t i t u t e s of Health (GM 24144), and we are a l s o grateful t o the Research Board of UIC f o r some f i n a n c i a l assistance.

Literature Cited 1. Kagan, J.; Kagan, E. D.; S e i g n e u r i e , E. Chemosphere 1986, 15, 49-57, and reference c i t e d . 2. Philogene, B. J . R.; Arnason, J . T. N a t i o n a l Meeting o f t h e Entomological Society o f America, Symposium on light-dependent interactions betwee Dec 12, 1984. 3. Kagan, J.; Kagan, ; , , 1984, 1115-1122. 4. Arnason, J . T.; Swain, T.; Wat, C. K.; Graham, E. A.; P a r t i n g t o n , S.; Lam, J.; Towers, G. H. N. Biochem. Syst. E c o l . 1981, 9, 63-68. 5. Wat, C. K.; Prasad, S.; Graham, E.; P a r t i n g t o n , S.; Arnason, T.; Towers, G. H. N.; Lam, J . Biochem. Syst. E c o l . 1981, 9, 5962. 6. Kagan, J.; Szczepanski, P.; Bindokas, V.; W u l f f , W. D.; McCallum, J . S. J . Chem. E c o l . 1986, 12, 899-914. 7. Kagan, J.; Kagan, E. D.; P a t e l , S.; P e r r i n e , D.; Bindokas, V. J . Chem. E c o l . 1987, 13, 593-604. 8. Tuveson, R. W.; Berenbaum, M. R.; H e i n i n g e r , E. E. J . Chem. E c o l . 1986, 12, 933-948. 9. Philogene, B. J . R.; Arnason, J . T.; Berg, C. W.; Duval, F.; Champagne, D.; T a y l o r , R. G.; L e i t c h , L.C.;Morand, P. J . Econom. Entomol. 1985, 78, 121-126. 10. Kagan, J . ; Chan, G. Experientia 1983, 39, 402-403. 11. Arnason, J . T.; Philogene, B. J . R.; Berg, C.; MacEachern, A.; Kaminski, J.; L e i t c h , L.C.;Morand, P.; Lam, J . Phytochem., 1986, 1609-1611. 12. Bennett, W. E.; Maas, J . L.; Sweeney, S. A.; Kagan, J . Chemosphere 1986, 15, 781-786. 13. Bowling, J . W.; Leversee, G. J.; Landrum, P. F.; Giesy, J . P. Aquat. T o x i c o l . 1983, 3, 79-90. 14. Reyftmann, J . P.; Kagan, J.; Santus, R.; M o r l i e r e , P. Photochem. Photobiol. 1985, 41, 1-7. 15. MacRae, W. D.; I r w i n , D. A. J.; B i s a l p u t r a , T.; Towers, G. H. N. Photobiochem. Photobiophys. 1980, 1, 309-318. 16. Foote, C. S. I n S i n g l e t Oxygen; Wasserman, H. H. and Murray, R. W., Eds.; Academic: New York, 1979; p 139. 17. Robinson, J . R.; Beatson, E. P. Pest. Biochem. Phys. 1985, 24, 375-383. 18. Philogene, B. J . R.; Arnason, J . T.; Berg, C. W.; Duval, F.; Morand, P. J. Chem. E c o l . 1986, 12, 893-898.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

12. 19. 20. 21. 22.

23.

KAGAN ET AL.

a-Terthienyl as a Photoactive Insecticide

191

Relyea, D. I.; Hubbard, W. L.; Grahame, J r . , R. E. U.S. P a t e n t 4,174,405, 1979. Relyea, D. I.; Moore, R. C.; Hubbard, W. L.; King, P. A. Proc. 10th Int. Congr. Plant Prot., V o l . 1, Croydon, U.K., p 355. Kagan, J.; Kagan, E. D., Kagan, P. A. Chemosphere 1985, 14, 1829-1834. Kagan, J.; Kagan, E. D., Kagan, P. A., I n A q u a t i c Photochemistry; Cooper, W. J.; Zika, R. G., Eds.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986; in press. Kagan, J . ; Kagan, E. D. Chemosphere 1986, 15, 243-251.

RECEIVED

December 22, 1986

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 13

Using Bacterial Mutants and Transforming DNA To Define Phototoxic Mechanisms R. W. Tuveson Department of Microbiology, University of Illinois, Urbana, IL 61801

Mutant Escherichi determine whether phototoxins damage cells by attacking DNA or cell membranes and to assess the a b i l i t y of phototoxins to induce excision repair and error-prone repair which leads to mutation. The r o l e of fatty acids versus e s s e n t i a l membrane proteins as potential l e t h a l targets for those phototoxins attacking the membrane can be evaluated using an E. c o l i fatty acid auxotroph. Inactivation of Haemophilus influenzae transforming DNA can evaluate DNA as a l e t h a l target for a phototoxin.

Microorganisms have played a central r o l e i n the development of current ideas concerning the mechanisms that underlie transmission of hereditary information, gene structure, and the regulation of gene function. Analyses i n v o l v i n g Escherichia c o l i and i t s viruses have been p a r t i c u l a r l y important i n these developments. E. c o l i i s probably the best understood system i n the b i o l o g i c a l world U_) p a r t l y because: 1) under optimal conditions, c e l l d i v i s i o n can occur every twenty (20) minutes, which provides f o r completion of experiments i n hours rather than days; 2) the minimal medium required for growth of w i l d type E. c o l i i s simple, consisting of s a l t s plus a carbon source (e.g., glucose); 3) E. c o l i i s a haploid organism; therefore, mutations are expressed d i r e c t l y without the need for complex matings to produce strains homozygous for induced mutations; 4) E. c o l l can be mated, and i n combination with generalized transduction i n v o l v i n g bacteriophage PI, sophisticated genetic analyses can be performed. Genetic manipulations are easy, and strains may be constructed for s p e c i f i c purposes (e.g., the study of the b i o l o g i c a l effects of phototoxins); 5) the techniques for cloning both eucaryotic and procaryotic genes i n v o l v e E. c o l i and i t s mutants.

0097-6156/87/0339-0192$06.00/0 © 1987 American Chemical Society

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

13.

TUVESON

E. c o l i —

Using Bacterial Mutants and Transforming DNA

193

DNA R e p a i r Systems

E. c o l i i s p a r t i c u l a r l y u s e f u l f o r the s t u d y o f p h o t o t o x i n s because i t s mechanisms f o r the r e p a i r o f r a d i a t i o n - and c h e m i c a l - i n d u c e d damage have been c h a r a c t e r i z e d i n d e t a i l ( 2 , 3). A minimum of s i x or seven E. c o l i DNA r e p a i r systems e x i s t , t h r e e o f w h i c h a r e c l e a r l y inducible. 1) P h o t o r e a c t i v a t i o n i s e f f e c t e d by an enzyme t h a t s p e c i f i c a l l y r e p a i r s i n t e r s t r a n d c y c l o b u t y l t h y m i n e dimers induced by far-UV (FUV; 200-300 nm). The p h o t o r e a c t i v a t i n g enzyme b i n d s t o dimers i n t h e dark and, upon exposure t o l i g h t ( r a n g i n g from 305-415 nm w i t h peak absorbance a t 320, 355, and 380 nm; 4 ) , the c o v a l e n t bonds between carbons 5 and 6 o f the a d j a c e n t dimers a r e e l i m i n a t e d i n s i t u , a l l o w i n g r e f o r m a t i o n of normal hydrogen bonding w i t h i n the DNA d o u b l e h e l i x . 2) E x c i s i o n r e p a i r i s a p r o c ess t h a t a c t u a l l y removes the l e s i o n from DNA. R e s y n t h e s i s f o l l o w s u s i n g t h e complementary, undamaged DNA s t r a n d as the t e m p l a t e . I n i t i a t i o n of t h i s r e p a i r e q u i r e i n c i s i o ste b complex s p e c i f i e d by t h endonuclease I ) . Polymeras the damaged s t r a n d d u r i n g t h e p o l y m e r i z a t i o n process. The new p a t c h o f DNA i s s e a l e d i n t o p l a c e by DNA l i g a s e . Kenyon and Walker (5) demonstrated t h a t a t l e a s t the product o f the u v r A gene i s i n d u c i b l e (one s u b u n i t o f the i n c i s i o n enzyme). 3) R e c o m b i n a t i o n a l or p o s t r e p l i c a t i o n a l r e p a i r i s p o s s i b l e because as polymerase I I I comes t o a l e s i o n (e.g., thymine d i m e r ) , i t s t a l l s f o r a few seconds and then moves beyond t h e dimer t o r e i n i t i a t e s y n t h e s i s . T h i s produces new (or daughter) DNA s t r a n d s c o n t a i n i n g gaps t h a t a r e r e p a i r e d by s i s t e r - s t r a n d exchange. These a r e f i l l e d by DNA polymerase I and f o l l o w e d by l i g a t i o n . C o n s e q u e n t l y , t h e DNA a r e a around the r e p l i c a t i o n f o r k i s a composite o f o l d DNA and n e w l y s y n t h e s i z e d DNA. T h i s i s n o t because DNA r e p l i c a t i o n i s c o n s e r v a t i v e , but because r e p a i r i s based on s i n g l e - s t r a n d replacement and r e s y n t h e s i s . 4) E r r o r - p r o n e r e p a i r i s induced by s p e c i f i c DNA-damaging agents. I t i s n o t c l e a r i f a new polymerase w i t h a reduced p r o o f - r e a d i n g f u n c t i o n i s i n d u c e d , o r i f t h e p r o o f - r e a d i n g f u n c t i o n o f polymerase I I I i s a l t e r e d i n some f a s h i o n . The outcome, however, i s t h a t p o l y m e r i z a t i o n can occur beyond l e s i o n s . T h i s r e s u l t s i n the i n s e r t i o n of n i t r o g e n o u s bases i n an a p p a r e n t l y random f a s h i o n . During the next round of r e p l i c a t i o n , a s t r a n d w i t h a new base p a i r i n the area o f the o r i g i n a l l e s i o n can r e s u l t ( i t may be expressed as a m u t a t i o n , depending on the base i n s e r t e d as the l e s i o n i s by-passed). A l t h o u g h the m o l e c u l a r d e t a i l s o f t h i s r e p a i r a r e not f u l l y under s t o o d , i t appears t h a t t h i s process i s r e s p o n s i b l e f o r the major f r a c t i o n o f induced m u t a t i o n . 5) I n the broadest sense, t h e "adap t i v e response" i n E. c o l i to e t h y l a t i n g and m e t h y l a t i n g agents can be c o n s i d e r e d as y e t another i n d u c i b l e E. c o l i r e p a i r system. P r e treatment o f E. c o l i w i t h e t h y l a t i n g or m e t h y l a t i n g agents (e.g., m e t h a n e s u l f o n a t e ) r e s u l t s i n r e s i s t a n c e t o t h e i n a c t i v a t i n g and mutagenic e f f e c t s o f these agents (6). I t has been shown b i o c h e m i c a l l y t h a t a t l e a s t two enzymes c a p a b l e o f removing a l k y l a t e d bases are induced i n response t o a l k y l a t i n g agents (3). The importance o f the " a d a p t i v e response" w i t h r e s p e c t to p h o t o t o x i n s i s q u e s t i o n a b l e . To our knowledge, no p h o t o t o x i n has been shown t o a l k y l a t e DNA. 6) E. c o l l possesses a s e r i e s o f enzymes t h a t r e c o g n i z e i n a p p r o p r i a t e n i t r o g e n o u s bases i n DNA and, t h e r e f o r e , a r e c a p a b l e o f removing

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

194

LIGHT-ACTIVATED PESTICIDES

them. T h i s c o r r e c t i o n system has been d e s c r i b e d as "mismatch r e p a i r . " O c c a s i o n a l l y , c y t o s i n e deaminates s p o n t a n e o u s l y y i e l d i n g u r a c i l . The enzyme u r a c i l N - g l y c o s y l a s e c l e a v e s the N - g l y c o s y l i c bond, a t t a c h i n g the i n a p p r o p r i a t e base to the DNA backbone. A second enzyme, a p u r i n i c (AP) e n d o n u c l e a s e , makes a s i n g l e - s t r a n d n i c k 5 t o the a p u r i n i c or a p y r i m i d i c s i t e , a l l o w i n g f o r t h e r e m o v a l of a few bases and r e s y n t h e s i s , u n d o u b t e d l y by polymerase I . Other g l y c o s y l a s e s e x i s t which r e c o g n i z e i n a p p r o p r i a t e bases i n DNA (e.g., hypoxanthine N - g l y c o s y l a s e ) . A l t h o u g h not y e t demonstrated, and perhaps important when c o n s i d e r i n g p h o t o s e n s i t i z e d t o x i c e f f e c t s , i t has been suggested t h a t g l y c o s y l a s e s s p e c i f i c f o r o x i d i z e d n i t r o g e nous bases e x i s t ( 3 , 7). 7) Demple and H a l b r o o k (8) r e c e n t l y have proposed t h a t E. c o l i has an i n d u c i b l e r e p a i r system s p e c i f i c f o r o x i d a t i v e damage independent o f those systems j u s t d e s c r i b e d . Since the l e t h a l i t y o f many p h o t o t o x i n s i s based on o x y g e n - r e l a t e d r a d i c a l s , such a r e p a i r system ( o r systems) w o u l d be p a r t i c u l a r l y impor t a n t to understand. U l t i m a t e l y i t may t u r n out t h a t t h i s p o s t u l a t e d system ( o r systems o x i d i z e d n i t r o g e n o u s base r e p o r t e d t h a t a t l e a s t t h i r t y p r o t e i n s i n E. c o l i a r e induced by " o x i d a t i v e s t r e s s " (9) and i t i s p o s s i b l e t h a t some o f these p r o t e i n s might be p a r t o f the p o s t u l a t e d o x i d a t i v e r e p a i r system(s). From t h i s b r i e f s u r v e y , i t i s apparent t h a t E. c o l i e x h i b i t s a m u l t i p l i c i t y o f systems f o r coping w i t h DNA damage. Therefore, u s i n g E. c o l i mutants d e f e c t i v e i n s p e c i f i c r e p a i r c a p a b i l i t i e s can p r o v i d e i n s i g h t s i n t o the mechanism(s) by which the c e l l s respond t o induced mutagenic and l e t h a l l e s i o n s . The s u b j e c t o f t h i s paper i s the use o f E. c o l i mutant s t r a i n s and Haemophilus i n f l u e n z a e t r a n s forming DNA (10) to study a s p e c t s o f the m u t a g e n i c i t y and l e t h a l i t y of p h o t o t o x i n s . f

E. c o l i RT7h-RT10h —

E x c i s i o n R e p a i r and C a t a l a s e

(HPII)

Ashwood-Smith et_ a l . (11) were among the f i r s t i n v e s t i g a t o r s t o use b a c t e r i a l mutants t o a i d i n c h a r a c t e r i z i n g p h o t o t o x i c e f f e c t s a t the m o l e c u l a r l e v e l . They demonstrated t h a t a F U V - s e n s i t i v e d e r i v a t i v e of E. c o l i B / r ( B ; 12) c o u l d be used to q u a n t i t a t i v e l y c h a r a c t e r i z e the e f f e c t s o f p a r t i c u l a r p h o t o t o x i n s (12). Because s t r a i n B _^ i s a d o u b l e mutant (13) i n which each m u t a t i o n c o n t r i b u t e s i n an a d d i t i v e way t o FUV s e n s i t i v i t y , i t cannot be deduced from e x p e r i ments i n v o l v i n g t h i s s t r a i n which o f the two d e f e c t s c a r r i e d by t h e s t r a i n , contributes to i t s s e n s i t i v i t y to a p a r t i c u l a r phototoxin. To c i r c u m v e n t t h i s p r o b l e m , we d e v e l o p e d a s e r i e s o f f o u r E. c o l i K12 s t r a i n s t h a t c a r r y a l l f o u r p o s s i b l e c o m b i n a t i o n s o f genes c o n t r o l l i n g e x c i s i o n p r o f i c i e n c y (uvrA6 versus u v r A ) and c a t a l a s e p r o f i c i e n c y ( k a t F v e r s u s k a t F [ f o r m e r l y d e s i g n a t e d as n u r v e r s u s nur ]; J ^ , 15). The u v r A a l l e l e s r a t h e r than the r e c A a l l e l e s were s e l e c t e d f o r i n c o r p o r a t i o n i n t o these s t r a i n s because: 1) The u v r A gene has been shown t o be i n d u c i b l e by s p e c i f i c DNA damaging agents (5) and i t i s r e p r e s e n t a t i v e o f t h e "SOS system o r r e g u l o n " (3). M u t a t i o n s i n the r e c A gene would e l i m i n a t e i n d u c t i o n o f any o f t h e many components o f the SOS r e g u l o n , i n c l u d i n g e r r o r - p r o n e r e p a i r n e c e s s a r y f o r an e v a l u a t i o n of mutagenesis by a p a r t i c u l a r photot o x i n . 2) Since r e c A m u t a t i o n s e l i m i n a t e r e c i p r o c a l r e c o m b i n a t i o n , i t would not be p o s s i b l e t o e a s i l y i n c o r p o r a t e new g e n e t i c markers s - 1

g

+

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195

i n t o these s t r a i n s i f t h i s proved n e c e s s a r y t o extend the i n v e s t i g a t i o n of a p a r t i c u l a r phototoxin i n a d i r e c t i o n not i n i t i a l l y a n t i c i pated. The a l l e l e s o f the u v r A gene permit e v a l u a t i o n of p h o t o t o x i n - i n d u c e d DNA l e s i o n s r e p a r a b l e by the e x c i s i o n r e p a i r system, w h i l e the k a t F a l l e l e s a l l o w f o r e v a l u a t i o n o f p h o t o t o x i n induced o x i d a t i v e damage ( p r o b a b l y from s u p e r o x i d e anion) s i n c e t h e k a t F a l l e l e s e n s i t i z e s c e l l s t o i n a c t i v a t i o n by hydrogen p e r o x i d e (16). In a d d i t i o n t o the u v r A and k a t F a l l e l e s , we have i n c o r p o r a t e d a r e v e r t i b l e h i s t i d i n e a l l e l e ( h i s - 4 ; 17) i n t o these s t r a i n s . T h i s a l l o w s e v a l u a t i o n o f mutagenesis by a p a r t i c u l a r p h o t o t o x i n i n the same experiments designed t o determine t h e n a t u r e o f the p h o t o t o x i c l e s i o n ( s ) . These s t r a i n s (RT7h, RT8h, RT9h, and RTlOh, T a b l e I ) Table I . E s c h e r i c h i a c o l i Bacterial Strains RT7h(tet ) L

Strains

F , thy-1 mtl-2 , m a l A l , s t r - 1 0 4 A X , supE44?

_

1

r

RT8h(tet )

r

same as R T 7 h ( t e t ) except uvrA

+

thi ,

+

r

RT9h(tet ) r

r

+

r

thi*

same as R T 7 h ( t e t ) except n u r

RT10h(tet )

same as R T 7 h ( t e t ) except

GW1060

F", t h r - 1 , l e u - 6 , h i s - 4 , a r g E 3 , i l v t s , t i f , s f i A l l , Alac(U169), g a l k 2 , s t r 3 1 , uvrA215::Mud(Aplac)

_5

K1060

F", fadE62, I a c I 6 0 , mel-1, supE57, supF58

27^, 30

have been t e s t e d w i t h p h o t o t o x i n s w i t h known mechanisms o f a c t i o n (18, 19), such as p s o r a l e n whose p h o t o t o x i c i t y i s a l m o s t e x c l u s i v e l y due t o c y c l o a d d i t i o n s to DNA. When the f o u r s t r a i n s were t r e a t e d w i t h p s o r a l e n p l u s near-UV (NUV; 300-400 nm), s t r a i n s RT7h and RT9h which c a r r i e d the uvrA6 a l l e l e proved t o be s e n s i t i v e t o i n a c t i v a t i o n ( F i g u r e 1). T h i s r e s u l t suggests t h a t p s o r a l e n l e s i o n s ( c y c l o a d d i t i o n s t o DNA) a r e r e p a r a b l e by t h e e x c i s i o n - r e p a i r system. The k a t F a l l e l e ( f o r m e r l y d e s i g n a t e d nur) does n o t s e n s i t i z e s t r a i n s t o p s o r a l e n p l u s NUV. T h i s c a n be i n t e r p r e t e d as e v i d e n c e t h a t oxygenr e l a t e d r a d i c a l s a r e not formed upon NUV treatment i n the presence of p s o r a l e n and i s c o n s i s t e n t w i t h t h e c h e m i c a l experiments i n v o l v ing p s o r a l e n p l u s NUV. Furthermore, i n these same e x p e r i m e n t s , we demonstrated, as e x p e c t e d , t h a t p s o r a l e n p l u s NUV treatment i s mutagenic, a l t h o u g h i t i s n o t as e f f i c i e n t a mutagen as i s FUV a l o n e (19). When the f o u r s t r a i n s were t r e a t e d w i t h a l p h a - t e r t h i e n y l (a-T), the k i n e t i c s o f i n a c t i v a t i o n were i n d i s t i n g u i s h a b l e ( F i g u r e 2). I n a d d i t i o n , h i s t i d i n e - i n d e p e n d e n t mutants were u n d e t e c t a b l e . A v a i l a b l e e v i d e n c e s u g g e s t s t h a t a-T a c t s as an oxygen-dependent

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

-5 10 J

1

1.8

NUV

1

1

1

1

3.6

5.4

7.2

9.0

( kilo joules rrf ) 2

F i g u r e 1. F l u e n c e - r e s p o n s e curve f o r f o u r E. c o l i s t r a i n s t r e a t e d w i t h broad-spectrum NUV i n the presence of p s o r a l e n . (Reproduced w i t h p e r m i s s i o n from Reference 19. C o p y r i g h t 1986 Plenum.)

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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F i g u r e 2. F l u e n c e - r e s p o n s e curves f o r f o u r E. c o l i s t r a i n s t r e a t e d w i t h broad-spectrum NUV i n the presence o f a - t e r t h i e n y 1 . (Reproduced w i t h p e r m i s s i o n from Reference 19. C o p y r i g h t 1986 Plenum.)

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(photodynamic) p h o t o s e n s i t i z e r (20) w i t h the p r i n c i p a l t a r g e t being the membrane (21) and our r e s u l t s are c o n s i s t e n t w i t h these c o n c l u s i o n s ( F i g u r e 2). The i n a c t i v a t i o n k i n e t i c s f o r the f o u r s t r a i n s are i n d i s t i n g u i s h a b l e and i m p l i e s t h a t the u v r A and k a t F a l l e l e s do not i n f l u e n c e the s e n s i t i v i t y of the c e l l s to i n a c t i v a t i o n by ot-T p l u s NUV. T h i s i s e x a c t l y what might be a n t i c i p a t e d i f ct-T d i d not e n t e r the c e l l to g e n e r a t e oxygen r a d i c a l s which a r e c a p a b l e of damaging DNA or o t h e r c e l l u l a r components i n the c y t o s o l . One would expect a-T p l u s near-UV to be non-mutagenic i f p e n e t r a t i o n i n t o the c e l l does not occur. The h a l f - l i f e of s i n g l e t oxygen ( O2) i s extended i n n o n - p o l a r environments (22) and the i n a c t i v a t i o n of the f o u r s t r a i n s may be caused by t h i s r e a c t i v e oxygen s p e c i e s t h a t damages e i t h e r the p r o t e i n s or the f a t t y a c i d components of the membrane (see the s e c t i o n e n t i t l e d : E. c o l i S t r a i n K1060 — F a t t y A c i d Auxotroph). In c o l l a b o r a t i o n w i t h J . Kagan, we used the f o u r E. c o l i s t r a i n s (RT7h-RT10h) t e v a l u a t th p h o t o t o x i c i t f non-carcino g e n i c , p o l y c y c l i c aromati found t h a t f l u o r a n t h e n s t r a i n s c a r r y i n g the uvrA6 m u t a t i o n were s e n s i t i v e to i n a c t i v a t i o n . In a d d i t i o n , the k a t F a l l e l e s e n s i t i z e d the c e l l s to i n a c t i v a t i o n by f l u o r a n t h e n e p l u s NUV, w h i c h i m p l i e d t h a t f l u o r a n t h e n e p l u s NUV g e n e r a t e s s u p e r o x i d e a n i o n , l e a d i n g to H2O2 and u l t i m a t e l y to the h y d r o x y l r a d i c a l (23). Chemical experiments have confirmed these c o n c l u s i o n s (manuscript submitted). We a l s o showed t h a t f l u o r anthene p l u s NUV treatment d i d not induce h i s t i d i n e - i n d e p e n d e n t m u t a t i o n s which suggested t h a t the treatment d i d not induce the e r r o r - p r o n e r e p a i r system known to be p a r t of the SOS r e g u l o n (3). S i n c e the u v r A gene i s p a r t of t h i s same r e g u l o n , we p r e d i c t e d t h a t treatment w i t h f l u o r a n t h e n e p l u s NUV would not induce the u v r A gene product (see s e c t i o n e n t i t l e d : E. c o l i S t r a i n GW1060 — Induction o f the uvrA Gene P r o d u c t ) • The experiments d e s c r i b e d above i l l u s t r a t e how the f o u r g e n e t i c a l l y d e f i n e d E. c o l l K12 s t r a i n s (RT7h-RT10h) can be used t o draw t e n t a t i v e c o n c l u s i o n s about the mechanism(s) of mutagenesis and l e t h a l i t y by p a r t i c u l a r p h o t o t o x i n s . These c o n c l u s i o n s may be used as g u i d e s f o r a d d i t i o n a l c h e m i c a l and b i o l o g i c a l experiments. E. c o l i S t r a i n GW1060 —

I n d u c t i o n of the u v r A Gene Product

Operon f u s i o n s t r a i n s p r o v i d e an e f f i c i e n t system f o r s t u d y i n g the r e g u l a t i o n o f l o c i whose gene p r o d u c t s are unknown o r d i f f i c u l t to assay. In p a r t i c u l a r , the Mud(Aplac) b a c t e r i o p h a g e , w i t h i t s appar e n t l y random i n t e g r a t i o n and e a s i l y assayed gene p r o d u c t , has been e x t r e m e l y u s e f u l i n these s t u d i e s of gene r e g u l a t i o n (24). Of the v a r i o u s Mud(Aplac) I n s e r t i o n s , GW1060, an E. c o l i s t r a i n w i t h a Mud(Aplac) i n s e r t i o n i n the u v r A l o c u s [uvrA215::Mud(Aplac)] , can be used t o demonstrate t h a t the e x p r e s s i o n of the u v r A gene i s i n d u c i b l e by DNA-damaging agents such as FUV and mitomycin C (5j F i g u r e 3A). In t h i s f u s i o n s t r a i n , 3 - g a l a c t o s i d a s e e x p r e s s i o n i s induced by these DNA-damaging agents i n a r e c A * jLex -dependent f a s h i o n (_5, 2 5 ) . We have t e s t e d three p h o t o t o x i n s f o r t h e i r a b i l i t y to induce the u v r A gene product u s i n g s t r a i n GW1060. To e s t a b l i s h t h a t the s t r a i n behaves i n our hands as I t d i d f o r Ken yon and Walker (_5), we +

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t r e a t e d the c e l l w i t h FUV and mitomycin C The c e l l s behaved e x a c t l y as d e s c r i b e d p r e v i o u s l y ( F i g u r e 3A; 5). We a n t i c i p a t e d t h a t p s o r a l e n p l u s NUV s h o u l d be c a p a b l e of i n d u c i n g the u v r A gene p r o d u c t s i n c e p s o r a l e n has been shown to form c y c l o a d d u c t s to DNA. The r e s u l t s d i s p l a y e d i n F i g u r e 3C s u b s t a n t i a t e t h i s p r e d i c t i o n . The i n d u c t i o n i s c l e a r l y the r e s u l t of the combination of p s o r a l e n p l u s NUV s i n c e n e i t h e r p s o r a l e n nor NUV a l o n e ( F i g u r e 3B and C) induce the u v r A gene product. Reported d a t a suggest t h a t a-T does not e n t e r c e l l s , but causes l e t h a l damage to the membrane (20, 21). Our r e s u l t s are t o t a l l y c o n s i s t e n t w i t h a-T o p e r a t i n g at the membrane l e v e l (19: see s e c tion entitled: E. c o l i S t r a i n s RT7h-RT10h — E x c i s i o n R e p a i r and C a t a l a s e (HPII)). We p r e d i c t e d c o r r e c t l y t h a t a-T p l u s NUV would not induce the u v r A gene product i f the membrane were the l e t h a l t a r g e t ( F i g u r e 3C). I n d u c t i o n of the u v r A gene product was not o b s e r v e d w i t h f l u o r a n t h e n e , a n o n - c a r c i n o g e n i c PAH p l u s NUV ( F i g u r e 3C). T h i s seems p a r a d o x i c a s e n s i t i z e d s t r a i n s RT7 p l u s NUV. It i s consistent, , f l u o r a n t h e n e p l u s NUV i s not c a p a b l e of i n d u c i n g h i s t i d i n e - i n d e p e n d ent m u t a t i o n s . Thus our i n t e r p r e t a t i o n i s t h a t f l u o r a n t h e n e p l u s NUV produces damage to DNA and the uninduced l e v e l of the u v r A gene product can r e p a i r these l e s i o n s . A l t e r n a t i v e l y , the damage p r o duced by f l u o r a n t h e n e p l u s near-UV does not form an i n d u c i n g s i g n a l n e c e s s a r y to i n c r e a s e l e v e l s of the u v r A gene product as w e l l as a l l o w i n g f o r m a t i o n of the e r r o r - p r o n e r e p a i r system which l e a d s to histidine-independent mutations. From the GW1060 r e s u l t s , one can suggest t h a t a-T p l u s NUV induces damage t h a t i s o x i d a t i v e s i n c e the r e s u l t s w i t h a-T p l u s NUV p a r a l l e l those o b t a i n e d w i t h H 0 . Hydro gen p e r o x i d e does not Induce h i s t i d i n e - i n d e p e n d e n t mutants i n s t r a i n s RT7h-RT10h (7) and i t does not induce the u v r A gene product ( F i g u r e 3B). I n c o n j u n c t i o n w i t h the i n a c t i v a t i o n experiments w i t h s t r a i n s RT7h-RT1Oh, s t r a i n GW1060 can be used to e s t a b l i s h whether a photo t o x i n i s c a p a b l e of producing a s i g n a l f o r the i n d u c t i o n of at l e a s t one component of the SOS r e g u l o n ( u v r A gene product). The r e s u l t s from these two e n t i r e l y d i f f e r e n t e x p e r i m e n t a l systems can be viewed as complementary and c o n f i r m a t o r y (e.g., a-T p l u s NUV i s not muta g e n i c [ f o r the h i s - 4 l o c u s a t l e a s t ] and i t does not induce e i t h e r the u v r A gene product or the e r r o r - p r o n e r e p a i r system, a p o r t i o n of the SOS r e g u l o n n e c e s s a r y f o r m u t a t i o n ) . The r e s u l t s from the experiments w i t h GW1060 and f l u o r a n t h e n e p l u s NUV p a r a l l e l the r e s u l t s o b t a i n e d when GW1060 and H 0 were used. T h i s i m p l i e s t h a t s u p e r o x i d e a n i o n i s an i m p o r t a n t product of f l u o r a n t h e n e p l u s NUV treatment. The c h e m i c a l r e s u l t s confirmed t h i s e x p e c t a t i o n (manu s c r i p t submitted). 2

2

E. c o l i K1060 —

2

2

F a t t y A c i d Auxotroph

E. c o l i s t r a i n K1060 can n e i t h e r s y n t h e s i z e nor degrade u n s a t u r a t e d f a t t y a c i d s ( f a t t y a c i d auxotroph). T h e r e f o r e , i t i s p o s s i b l e to c o n t r o l the amount and type of u n s a t u r a t e d f a t t y a c i d i n the mem brane by growing the c e l l s i n medium of a p a r t i c u l a r c o m p o s i t i o n supplemented w i t h the a p p r o p r i a t e f a t t y a c i d (26, 27). In a d d i t i o n to DNA and s o l u b l e p r o t e i n s , p h o t o t o x i n s (e.g., a-T; 21) may a t t a c k

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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201

the p r o t e i n s or the f a t t y a c i d s of the membrane. Because the h a l f l i f e of 0 i s extended i n n o n - p o l a r e n v i r o n m e n t s (22) and because 0 r e a c t s w i t h u n s a t u r a t e d f a t t y a c i d d o u b l e bonds (28, 29), oxy gen-dependent p h o t o t o x i n s t h a t do not have DNA as an important l e t h a l t a r g e t p r o b a b l y generate 0 t h a t r e a c t s w i t h the u n s a t u r a t e d f a t t y a c i d s o f the membrane d e s t r o y i n g i t s h y d r o p h o b i c ! t y l e a d i n g to g e n e r a l i z e d membrane d i s i n t e g r a t i o n . I f t h i s i s t r u e , we s h o u l d be a b l e to t e s t t h i s p o s s i b i l i t y by growing E. c o l i s t r a i n K1060 i n the presence of f a t t y a c i d s w i t h v a r y i n g degrees of u n s a t u r a t i o n . The p r a c t i c a l i t y o f t h i s approach was i l l u s t r a t e d by experiments w i t h s t r a i n K1060 t h a t was grown f i r s t w i t h twenty carbon f a t t y a c i d s ( e i c o s a e n o i c a c i d d e r i v a t i v e s ) e x h i b i t i n g v a r i o u s degrees of u n s a t u r a t i o n ( 1 , 2, 3, or 4) and then t r e a t e d w i t h NUV. These e x p e r i m e n t s showed t h a t the s e n s i t i v i t y to i n a c t i v a t i o n by NUV o f the v a r i o u s e x p o n e n t i a l l y growing p o p u l a t i o n s was r e l a t e d to the degree of u n s a t u r a t i o n o f the f a t t y a c i d s used f o r s u p p l e m e n t a t i o n . The g r e a t e r the degree of u n s a t u r a t i o n the c e l l s to i n a c t i v a t i o endogenous p h o t o s e n s i t i z e r s f o NU col c e l l s are the c y t o chromes l o c a t e d w i t h i n the membrane (15). I t seems l i k e l y t h a t a b s o r p t i o n of NUV w a v e l e n g t h s by cytochromes g e n e r a t e s 0 w h i c h has the p o t e n t i a l f o r r e a c t i n g w i t h the u n s a t u r a t e d f a t t y a c i d s w i t h i n the membrane. T h i s would account f o r the l e t h a l i t y and r e l a t i v e n o n - m u t a b i l i t y o f these w a v e l e n g t h s . I f the r e s u l t s r e p o r t e d by Downum e t a l . (21) s u g g e s t i n g t h a t the l e t h a l t a r g e t s f o r i n a c t i v a t i o n by a-T p l u s NUV are membrane p r o t e i n s are c o r r e c t , c e l l s of s t r a i n K1060 grown w i t h f a t t y a c i d s h a v i n g v a r i o u s degrees of unsa t u r a t i o n s h o u l d be e q u i v a l e n t i n t h e i r s e n s i t i v i t y to i n a c t i v a t i o n by a-T p l u s NUV. The r e s u l t s of e x p e r i m e n t s designed to t e s t t h i s p r e d i c t i o n are presented i n F i g u r e 4. I f the f a t t y a c i d s of the membrane were an important l e t h a l t a r g e t , s t r a i n K1060 grown w i t h a r a c h i d o n i c a c i d (20°C, 4 d o u b l e bonds) s u p p l e m e n t a t i o n s h o u l d have been s e n s i t i v e to i n a c t i v a t i o n by a-T p l u s NUV. C l e a r l y , these c e l l s were no more s e n s i t i v e to i n a c t i v a t i o n by a-T p l u s NUV than were c e l l s grown w i t h 1 1 - e i c o s a e n o i c a c i d s u p p l e m e n t a t i o n (20°C, 1 d o u b l e bond). We c o n c l u d e from these r e s u l t s t h a t the l e t h a l i t y of a-T p l u s NUV t o E. c o l i c e l l s r e s u l t s from damage to membrane p r o t e i n s , as suggested by Downum e t a l . (21). I t s h o u l d be noted t h a t the NUV f l u e n c e s used i n these experiments do not r e s u l t i n s i g n i f i cant i n a c t i v a t i o n u n l e s s a-T i s present. S t r a i n K1060 can be used w i t h p h o t o t o x i n s , the l e t h a l t a r g e t of which appears to be the membrane. I t can be used t o d i f f e r e n t i a t e between the u n s a t u r a t e d f a t t y a c i d s of the membrane and the e s s e n t i a l membrane p r o t e i n s as the l e t h a l t a r g e t s . 2

2

2

2

E. c o l i HB101

—

Cloned Carotenoid

Genes

R e c e n t l y , P r o f e s s o r J e f f r e y L. Bennetzen's group (Department of B i o l o g y , Purdue U n i v e r s i t y ) has c l o n e d c a r o t e n o i d genes from E. s t e w a r t i i ( t h e i n c i t a n t o f corn w i l t ) i n t o E. c o l i s t r a i n HB101, and they have o b t a i n e d e x p r e s s i o n of these genes. P r o f e s s o r Bennetzen has p r o v i d e d us w i t h the s t r a i n s i n which c a r o t e n o i d s are expressed and the HB101 s t r a i n i n t o which the c a r o t e n o i d genes were c l o n e d . S i n c e c a r o t e n o i d s are p a r t of the membrane, the c a r o t e n o i d - p r o d u c i n g E. c o l i s t r a i n s can be used to t e s t whether c a r o t e n o i d s p r o t e c t the

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

LIGHT-ACTIVATED PESTICIDES

F i g u r e 4. F l u e n c e - r e s p o n s e c u r v e s f o r e x p o n e n t i a l l y growing and s t a t i o n a r y c e l l p o p u l a t i o n s o f E. c o l i s t r a i n K1060 when grown w i t h v a r i o u s f a t t y a c i d s as supplements.

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c e l l a g a i n s t i n a c t i v a t i o n by a p a r t i c u l a r p h o t o t o x i n . I f p r o t e c t i o n i s o b s e r v e d , one c o u l d p o s t u l a t e t h a t the p r i n c i p a l l e t h a l e v e n t s occur w i t h i n the membrane and t h a t s i n g l e t oxygen may be i n v o l v e d . These r e s u l t s would complement those o b t a i n e d w i t h E. c o l l s t r a i n K1060 ( f a t t y a c i d auxotroph). A l t h o u g h i n v e s t i g a t i o n s w i t h the c a r o t e n o i d and c a r o t e n o i d l e s s E. c o l i s t r a i n s a r e i n t h e e a r l y s t a g e s , we have shown t h a t the c a r o t e n o i d - p r o d u c i n g s t r a i n s a r e p r o t e c t e d a g a i n s t the i n a c t i v a t i n g e f f e c t s o f a 3 - c a r b o l i n e a l k a l o i d (harmine) p l u s NUV as w e l l as NUV a l o n e . The c a r o t e n o i d and c a r o t e n o i d l e s s E. c o l i s t r a i n s h o l d g r e a t promise as p o s s i b l e t o o l s t o a i d i n the b i o l o g i c a l i n v e s t i g a t i o n s o f p h o t o t o x i n s . Transforming

DNA —

Haemophilus i n f l u e n z a e

Transforming DNA i s a r e l a t i v e l y s i m p l e and d i r e c t method f o r t e s t i n g the e f f e c t s o f p h o t o t o x i n s on t h e b i o l o g i c a l a c t i v i t y o f DNA, and t r a n s f o r m i n g DN the system o f c h o i c e . C e l l b r i e f p e r i o d of a n a e r o b i o s i s and can be s t o r e d a t -80°C f o r months w i t h o n l y a modest l o s s i n competence. W i t h l i t t l e d i f f i c u l t y , t r a n s f o r m i n g DNA from whole c e l l s can be prepared by s t a n d a r d p r o c e dures. P l a t i n g s to assay f o r t r a n s f o r m a t i o n can be done by pour p l a t i n g s i n c e t h i s R. i n f l u e n z a e i s n o t an o b l i g a t e aerobe, as i s B a c i l l u s s u b t i l i s . I n a d d i t i o n , p o u r - p l a t i n g p e r m i t s c o u n t i n g hun dreds o f c o l o n i e s w i t h l i t t l e d i f f i c u l t y . We have used H. i n f l u e n z a e t r a n s f o r m i n g DNA t o t e s t the e f f i c a c y o f v a r i o u s p h o t o t o x i n s f o r the i n a c t i v a t i o n o f t r a n s f o r m i n g a c t i v i t y . The g e n e r a l r e s u l t i s t h a t p h o t o t o x i n s t h a t form c y c l o a d d i t i o n s to DNA (e.g., p s o r a l e n and a n g e l i c i n ) are h i g h l y e f f i c i e n t for the i n a c t i v a t i o n o f t r a n s f o r m i n g a c t i v i t y . P h o t o t o x i n s t h a t appear to a c t c h i e f l y by t h e g e n e r a t i o n of oxygen r a d i c a l s p e c i e s (a-T and f l u o r a n t h e n e ) a r e much l e s s e f f i c i e n t . When comparing p s o r a l e n (0.2 ug m l ) w i t h f l u o r a n t h e n e (10.0 ug m l ), f o r exam p l e , the f l u e n c e r e q u i r e d t o reduce t r a n s f o r m i n g a c t i v i t y t o 0.37 was 1 5 - f o l d g r e a t e r w i t h f l u o r a n t h e n e (manuscript s u b m i t t e d ) . Our i m p r e s s i o n i s t h a t the p h o t o t o x i n s we have i n v e s t i g a t e d separate i n t o two broad c a t e g o r i e s w i t h r e s p e c t to i n a c t i v a t i o n of t r a n s forming a c t i v i t y , those t h a t c o v a l e n t l y b i n d to DNA, and those t h a t form oxygen-re l a ted r a d i c a l s . The l a t t e r a r e r e l a t i v e l y i n e f f e c t i v e i n r e d u c i n g t r a n s f o r m i n g a c t i v i t y and seem t o be s t r o n g l y i n f l u e n c e d by the s o l v e n t i n which the p h o t o t o x i n i s d i s s o l v e d ( p r e s u m a b l y r e f l e c t i n g excimer f o r m a t i o n ) . We a r e c u r r e n t l y t e s t i n g t h i s p o s s i bility. Conclusions The b i o l o g i c a l a c t i v i t y o f suspected p h o t o t o x i n s can be assessed I n a m a t t e r o f days ( p r o b a b l y two working weeks once the systems a r e i n p l a c e ) by u s i n g the b a c t e r i a l systems d e s c r i b e d i n t h i s paper. M i c r o b i a l systems a r e easy t o use and r e s u l t s can be o b t a i n e d q u i c k l y . These b i o l o g i c a l experiments, i n c o n j u n c t i o n w i t h the a p p r o p r i a t e c h e m i c a l experiments, can r e s u l t i n an a c c u r a t e p i c t u r e of the mode o f a c t i o n o f a p a r t i c u l a r p h o t o t o x i n b e f o r e the much more l a b o r i o u s t e s t s i n v o l v i n g e u c a r y o t i c b i o l o g i c a l systems are undertaken. Determining the mechanism of a c t i o n of a p h o t o t o x i n

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u s i n g m i c r o b i a l systems p r o v i d e s p l a u s i b l e e x p l a n a t i o n s f o r r e s u l t s o b t a i n e d w i t h e u c a r y o t i c systems i n w h i c h mutants a r e u s u a l l y n o t available. Acknowledgments T h i s r e s e a r c h was supported by a g r a n t from the N a t i o n a l S c i e n c e F o u n d a t i o n (PCM 8315595). The a u t h o r w i s h e s t o e x p r e s s h i s s i n c e r e a p p r e c i a t i o n t o Ms. Nancy Bode f o r h e r i n v a l u a b l e h e l p i n the p r e p a r a t i o n o f t h i s manuscript. Literature Cited 1. Bachman, B. J. M i c r o b i o l . Rev. 1983, 47, 180-230. 2. Witkin, E. M. B a c t e r i o l . Rev. 1976, 40, 869-907. 3. Walker, G. C. M i c r o b i o l . Rev. 1984, 48, 60-93. 4. Jagger, J. Photochem Photobiol 1964 3 451—61 5. Kenyon, C. J.; Walker 10. 6. Samson, L.; C a r i n s , J. Nature (London) 1977,267,281-2. 7. Sammartano, L. J.; Tuveson, R. W. Photochem. Photobiol. 1985, 41, 367-70. 8. Demple, B.; Halbrook, J. Nature (London) 1983, 30, 466-8. 9. Christman, M. F.; Morgan, R. W.; Jacobson, F. S.; Ames, B. N. Cell 1985, 41, 753-62. 10. Setlow, J. K.; Brown, D.C.;B o l i n g , M. E.; M a t t i n g l y , A.; Gordon, M. P. J . Bact. 1968, 95, 546-58. 11. Ashwood-Smith, M. J.; P o u l t o n , G. A.; Ceska, O.; L i e u , M.; Furniss, E. Photochem. Photobiol. 1983, 38, 113-8. 12. Hill, R. F.; Simson, E. J . Gen. M i c r o b i o l . 1961, 24, 1-14. 13. Greenberg, J . Genetics 1967, 55, 193-201. 14. Leonardo, J. M.; Reynolds, P. R.; Tuveson, R. W. Mutat. Res. 1984, 126, 1-8. 15. Tuveson, R. W.; Sammartano, L. J. Photochem. Photobiol. 1986, 42, 621-6. 16. Sammartano, L. J.; Tuveson, R. W.; Davenport, R. J . Bact. 1986, 168, 13-21. 17. Kato, T.; Rothman, R. H.; C l a r k , A. J. Genetics 1977, 87, 118. 18. S c o t t , B. R.; Pathak, M. A.; Mohn, G. R. Mutat. Res. 1976, 39, 29-74. 19. Tuveson, R. W.; Berenbaum, M. R.; H e i n i n g e r , E. E. J . Chem. E c o l . 1986, 12, 933-48. 20. Arnason, T.; Chang, G. F. Q.; Wat, C. K.; Downum, K.; Towers, G. H. N. Photochem. Photobiol. 1981, 38, 811-24. 21. Downum, K. R.; Hancock, R. W. E.; Towers, G. H. N. Photochem. Photobiol. 1982, 36, 517-23. 22. Korycka-Dahl, M. B.; Richardson, T. In CRC C r i t i c a l Reviews i n Food Science and Nutrition; Furia, T. E., Ed.; CRC Press, Inc.: West Palm Beach, FL, 1978; pp 209-38. 23. Larson, R. A. J . Chem. E c o l . 1986, 12, 859-70. 24. Casadaban, M. J.; Cohen, S. N. Proc. N a t l . Acad. S c i . (USA) 1979, 76, 4530-33. 25. Kenyon, C. J.; Walker, G. C. Proc. N a t l . Acad. S c i . (USA) 1980, 77, 2819-23.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

13.

TUVESON

26. 27. 28. 29. 30. 31.

Using Bacterial Mutants and Transforming DNA

205

T a y l o r , F.; Cronan, J. E. J . Bact. 1976, 125, 518-23. Redpath, J. L.; P a t t e r s o n , L. K. Radiat. Res. 1978, 75, 443-7. Hall, G. E.; Roberts, D. G. J . Chem. Soc.(B)1966,11,110912. Cobern, D.; Hobbs, J . S.; Lucas, R. A.; MacKenzie, D. J. J. Chem. Soc. (C) 1966, 21, 1897-1902. Klamen, D. L.; Tuveson, R. W. Photochem. P h o t o b i o l . 1982, 35, 167-73. Tuveson, R. W. Genet. Res. (Camb.) 1972, 20, 9-18.

R E C E I V E D November 20, 1986

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 14

Charge of the Light Brigade: Phototoxicityasa Defense Against Insects M. R. Berenbaum Department of Entomology, University of Illinois, Urbana, IL 61801-3795

Sunlight i s used by many plants to activate secondary compounds and to enhanc a c t i v a t i o n can occu be absorbed by plan , furanocoumarins t y p i c a l of the Umbelliferae and Rutaceae, to a l t e r the electron configuration and form a highly reactive excited state; the excited state molecule can then interact d i r e c t l y with biomolecules such as DNA, proteins or membrane l i p i d s with concomitant toxic effects. A l t e r n a t i v e l y , as i s the case for polyacetylenes t y p i c a l of the Compositae and quinones of the Guttiferae, photopromoted excited states can interact with oxygen to form the reactive molecule s i n g l e t oxygen, which then can i n t e r f e r e chemically with other biomolecules. Toxicity enhancement by sunlight i s increased still further by v i r t u e of the fact that certain wavelengths can stimulate enhanced biosynthesis and increased accumulation of phototoxins. N a t u r a l l y occurring phototoxins occur i n a diverse array of plant f a m i l i e s and represent a v a r i e t y of b i o s y n t h e t i c a l l y unrelated structures. Many of these chemicals are toxic to generalized feeders , p a r t i c u l a r l y i n the presence of l i g h t of the appropriate wavelengths. E s s e n t i a l l y every phototoxic plant i s associated with oligophagous species which have overcome the defensive chemistry of their hosts. Mechanisms of resistance include behavioral resistance i n the form of l e a f - r o l l i n g , web-spinning, and other forms of concealed feeding which s h i e l d the insect from damaging wavelengths, physical resistance i n the form of body pigments that s e l e c t i v e l y absorb damaging wavelengths or quench excited states, or biochemical resistance i n the form of enzymatic degradation of phototoxic molecules. Sunlight, then, i s an important e c o l o g i c a l factor mediating the evolutionary responses between plants and herbivorous insects.

0097-6156/87/0339-0206$06.00/0 © 1987 American Chemical Society

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14.

BERENBAUM

Phototoxicity as a Defense Against Insects

207

The concept of u s i n g l i g h t energy f o r d e f e n s i v e purposes, ( v i z , the " S t r a t e g i c Defense I n i t i a t i v e " of the Reagan a d m i n i s t r a t i o n ) i s h a r d l y an i n n o v a t i o n ; p l a n t s have i n c o r p o r a t e d s u n l i g h t i n t o t h e i r d e f e n s i v e armamentarium f o r m i l l e n i a (1). Many p l a n t a l l e l o c h e m i c a l s can absorb photons of l i g h t energy at p a r t i c u l a r w a v e l e n g t h s . T h i s energy can t r a n s f o r m a m o l e c u l e from i t s l o w e s t e l e c t r o n energy s t a t e or ground s t a t e to a h i g h e r or e x c i t e d s t a t e . These e x c i t e d s t a t e s are h i g h l y r e a c t i v e and p h o t o t o x i c p l a n t c h e m i c a l s can r e a c t w i t h a v a r i e t y of b i o m o l e c u l e s . In the case of f u r a n o c o u m a r i n s , f o r example, compounds t y p i c a l l y found In p l a n t s i n the f a m i l i e s Rutaceae and Umbel 1 i f e r a e , e x c i t e d t r i p l e t s r e a c t w i t h p y r i m i d i n e bases i n DNA to form c y c l o a d d u c t s t h a t i m p a i r t r a n s c r i p t i o n and r e p l i c a t i o n . In many o t h e r p h o t o s e n s i t i z e r s , the e x c i t e d t r i p l e t s t a t e r e a c t w i t h m o l e c u l a r oxygen, which i n i t s ground s t a t e i s a t r i p l e t . The s i n g l e t oxygen t h a t r e s u l t s i s h i g h l y r e a c t i v e and can damage p r o t e i n s , l i p i d s and DNA. Ground s t a t e oxygen can a l s o for p h o t o s e n s i t i z e r ; these p o l y s a c c h a r i d e s (1). S i n c e the t a r g e t s i t e s f o r p h o t o s e n s i t i z i n g compounds are o f t e n important b i o m o l e c u l e s , n a t u r a l p h o t o s e n s i t i z e r s are b r o a d l y b i o c i d a l . However, i t has l o n g been r e c o g n i z e d (2) t h a t h e r b i v o r o u s i n s e c t s , as a major s e l e c t i v e f o r c e on p l a n t s , are l i k e l y to be a p r i n c i p a l m o t i v e f o r c e behind the e v o l u t i o n a r y p r o l i f e r a t i o n of t o x i c c h e m i c a l s In p l a n t t i s s u e . Such i s l i k e l y the case f o r p h o t o s e n s i t i z e r s as w e l l . N a t u r a l p h o t o t o x i n s were f i r s t shown to have i n s e c t i c i d a l p r o p e r t i e s i n 1978 (3); s i n c e t h a t time, a t l e a s t n i n e b i o s y n t h e t i c a l l y d i s t i n c t c l a s s e s of p h o t o t o x i c i n s e c t i c i d e s have been i d e n t i f i e d ( T a b l e I ) . S u n l i g h t , then, can a c t at the c h e m i c a l l e v e l , enhancing the t o x i c i t y of d e f e n s i v e c h e m i c a l s s y n t h e s i z e d by p l a n t s . S u n l i g h t can a l s o a f f e c t m e t a b o l i c r a t e s i n p l a n t s ; i n c r e a s i n g UV l i g h t i n t e n s i t y s e l e c t i v e l y s t i m u l a t e s enzyme a c t i v i t y and enhances b i o s y n t h e t i c r a t e s f o r a v a r i e t y o f n a t u r a l p r o d u c t s ( T a b l e I I ) . I n d u c t i o n of p h e n y l a l a n i n e ammonia l y a s e by u l t r a v i o l e t i r r a d i a t i o n , e.g., d i r e c t l y a f f e c t s p r o d u c t i o n of p h e n y l p r o p a n o i d s , coumarins, f l a v o n o i d s , acetophenones and l i g n a n s (Berenbaum 1987, i n p r e s s ) . M o r e o v e r , s u n l i g h t can a c t as an i n d i r e c t f a c t o r i n f l u e n c i n g the c h e m i c a l p r o f i l e of a p l a n t s p e c i e s . In t h a t w a v e l e n g t h and i n t e n s i t y are two f a c t o r s i n f l u e n c i n g p h o t o s y n t h e t i c r a t e s of p l a n t s , they can a l s o i n f l u e n c e a l l e l o c h e m i c a l p r o d u c t i o n i n those i n s t a n c e s i n w h i c h b i o s y n t h e s i s i s e n e r g y - l i m i t e d . Enhanced p h o t o s y n t h e t i c r a t e s p r o v i d e more energy to c h a n n e l i n t o b i o s y n t h e s i s (4).

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Table I . P l a n t d e r i v e d - p h o t o t o x i n s w i t h i n s e c t i c i d a l Class Acetylenes Benzopyrans and f u r a n s Benzylisoquinoline alkaloids Beta-carboline alkaloids Extended quinones Furanocoumarins

Furanochromones Furoquinoline alkaloids Thiophenes

Table I I .

properties

Reference 5, 6, 7

Source Compositae

A r e g u l l i n , t h i s volume Compositae B e r b e r i d a c e a e , Rutaceae, 8 Rubiaceae Rutaceae, Simaroubaceae 9, E. H e i n i n g e r , i n prep. G u t t i f e r a e , Polygonaceae 10 Leguminosae 3 11 12 1 3 14 Moraceae Umbelliferae Solanaceae Rutaceae, U m b e l l i f erae 11, 15 Rutaceae 9, E. H e i n i n g e r , i n prep. Compositae 5, 7, 16, 17

P l a n t compounds induced o r i n c r e a s e d by l i g h t

Plant compound

L i g h t source

P l a n t source

Ref

Alkaloids Alkaloids Anthocyanins Betacyanins Cannabinoids Cardenolides Carotenoids DIMBOA Flavonoids Furanocoumarins

red and IR Visible Visible red UV Visible blue l i g h t Visible UV UV

tobacco lupines,tobacco many p l a n t s Centrospermae marijuana D i g i t a l i s lanata many s p e c i e s Zea mays Umbelliferae parsnip

Isoflavonoids Tannins Terpenes

UV "sunlight" "sunlight"

soybean oak Hymenaea courbaril

18 18 19 19 20 21 22 23 24 Berenbaum and Z a n g e r l 1987, i n press 25 26 27

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E f f i c a c y o f p h o t o t o x i c i t y as a defense a g a i n s t i n s e c t s No defense system i s u n b r e a c h a b l e , and l i g h t - d e p e n d e n t defense systems o f p l a n t s a r e no e x c e p t i o n . C o n t i n u i n g s e l e c t i o n by p l a n t c h e m i c a l s promotes the a c q u i s i t i o n o f r e s i s t a n c e by h e r b i v o r o u s i n s e c t s o v e r e v o l u t i o n a r y time. However, s e l e c t i o n by p h o t o t o x i n s d i f f e r s from the s t a n d a r d s c e n a r i o (e.g., 28) i n t h a t the s e l e c t i v e f o r c e promoting the e v o l u t i o n o f r e s i s t a n c e can e i t h e r be the c h e m i c a l i t s e l f o r the s u n l i g h t c o n f e r r i n g t o x i c i t y to the c h e m i c a l . Insect associates of phototoxic p l a n t s d i s p l a y a v a r i e t y of a d a p t a t i o n s t o p h o t o t o x i c p l a n t s t h a t e i t h e r reduce the c h e m i c a l r e a c t i v i t y o r p h y s i o l o g i c a l e f f e c t s o f the substances i n v o l v e d o r m i n i m i z e t h e i r exposure t o l e t h a l amounts o r w a v e l e n g t h s o f sunlight. Behavioral resistance Behavioral resistance f a i l u r e o f an i n s e c t to i n g e s t o r c o n t a c t a l e t h a l dose o f t o x i c a n t o r from the a b i l i t y o f an i n s e c t t o feed i n such a manner as t o reduce the amount o f l i g h t exposure b e l o w t h a t r e q u i r e d to a c t i v a t e a p h o t o t o x i n . Feeding i n a c o n c e a l e d manner i s c h a r a c t e r i s t i c o f a number o f i n s e c t a s s o c i a t e s o f a number o f p h o t o t o x i c p l a n t s . Modes of concealed feeding i n c l u d e l e a f mining, l e a f t y i n g , l e a f r o l l i n g , stem b o r i n g , s u b t e r r a n e a n r o o t f e e d i n g , o r b o r i n g i n t o buds o r f r u i t s . I n a l l these c a s e s , p l a n t t i s s u e s can e f f e c t i v e l y b l o c k s i g n i f i c a n t amounts o f damaging s u n l i g h t . For example, i n a s u r v e y of l e a f e p i d e r m a l t r a n s m i t t a n c e o f UV r a d i a t i o n i n 25 s p e c i e s o f p l a n t s , t r a n s m i t t a n c e i n most cases was l e s s than 10% and i n o v e r h a l f the s p e c i e s ranged from 1 to 5% (29). B e h a v i o r a l a v o i d a n c e o f p h o t o t o x i n s i s a widespread phenomenon; o v e r 70% o f the fauna o f p h o t o t o x i c U m b e l l i f erae i n one study c o n s i s t e d of i n s e c t s f e e d i n g i n a c o n c e a l e d manner (3). Concealed f e e d e r s can e i t h e r be h i g h l y s p e c i a l i z e d (as i s the case f o r umbel l i f e r - f e e d i n g l e a f - m i n i n g Agromyzidae) o r b r o a d l y polyphagous (as i s the case f o r C h o r i s t o n e u r a rosaceana, a t o r t r i c i d l e a f r o l l e r t h a t feeds on a number of p h o t o t o x i c p l a n t s i n the U m b e l l i f erae and i n s e v e r a l o t h e r families). Champagne e t a l . 1986 suggest t h a t , i n a d d i t i o n t o b o r i n g i n t o stems, s p i n n i n g p r o f u s e amounts of s i l k a l s o s e r v e s t o a t t e n u a t e incoming r a d i a t i o n and to p r o t e c t O s t r i n i a n u b i l a l i s , the European c o r n b o r e r , from p h o t o t o x i c a c e t y l e n e s i n i t s h o s t s i n the f a m i l y Compositae. The most c o m p e l l i n g e v i d e n c e f o r a p h o t o p r o t e c t i v e r o l e f o r c o n c e a l e d f e e d i n g i n v o l v e s the i n s e c t fauna o f Hypericum p e r f o r a t u m , St. Johnswort o r Klamath weed. JL^ p e r f o r a t u m c o n t a i n s an extended quinone pigment, h y p e r i c i n , which i s a c t i v a t e d by s u n l i g h t i n t h e r e g i o n o f 500-600 nm (30). A l t h o u g h a p r o p o r t i o n o f the fauna o f St. Johnswort c o n s i s t s o f s p e c i a l i s t s , t h e r e a r e s e v e r a l b r o a d l y polyphagous s p e c i e s t h a t can p r e d i c t a b l y be found f e e d i n g on the f o l i a g e and f l o w e r s ( T a b l e I I I ) . Of t h e s e , one s p e c i e s (a l y c a e n i d c a t e r p i l l a r ) bores i n t o f l o w e r s and f r u i t s , and f i v e ( a l l t o r t r i c i d c a t e r p i l l a r s ) t i e t o g e t h e r l e a v e s , stems o r f l o w e r s and feed i n s i d e these t i e s . O s t e n s i b l y , s i n c e f o l i a g e i s e s s e n t i a l l y opaque t o most w a v e l e n g t h s o f l i g h t , c a t e r p i l l a r s c o n c e a l e d i n l e a f t i e s can feed

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

210

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on p h o t o t o x i c m a t e r i a l w i t h i m p u n i t y ; i n s u f f i c i e n t amounts o f l i g h t p e n e t r a t e to a c t i v a t e the p h o t o t o x i n . When one o f the t o r t r i c i d s , P l a t y n o t a f l a v e d a n a , I s r e a r e d i n the l a b o r a t o r y on an a r t i f i c i a l d i e t , i t cannot engage i n l e a f - t y i n g b e h a v i o r and i s f o r c e d t o feed i n an manner such t h a t i t i s exposed t o l i g h t . C a t e r p i l l a r s r e a r e d on an a r t i f i c i a l d i e t c o n t a i n i n g h y p e r i c i n s u f f e r e d s i g n i f i c a n t l y g r e a t e r m o r t a l i t y when exposed t o f u l l s u n l i g h t than when they were p r o t e c t e d from damaging w a v e l e n g t h s by an a c e t a t e f i l t e r ( T a b l e I V S. Sandberg, i n prep.). L e a f - t y i n g may thus be a p r e a d a p t a t i o n a l l o w i n g g e n e r a l i z e d f e e d e r s , l a c k i n g a s p e c i f i c d e t o x i c a t i o n system f o r p h o t o t o x i n s , t o e x p l o i t p h o t o t o x i c p l a n t s . Such b e h a v i o r may i n f a c t be f a c u l t a t i v e , when P. f l a v e d a n a feeds on the f o l i a g e o f strawberry (Fragaria v i r g i n i e n s i s ) , a nonphototoxic p l a n t , i t o c c a s i o n a l l y spins only a s c a f f o l d i n g of s i l k i n place of a l e a f f o l d i n i t s e a r l y i n s t a r s . Of the r e m a i n i n g l e p i d o p t e r o u s a s s o c i a t e s o f Hypericum, s p e c i e s i n the n o c t u i d genus P o l l a feed n o c t u r n a l l y (G. G o d f r e y are m i n i m i z e d . N o c t u r n a l l preadapted f o r f e e d i n g on p h o t o t o x i c p l a n t s . Table I I I . Lepldopteran associates of Hypericum Species

Family

Strymon me11 mis Zale lunata Polla assimllls

31 f l o w e r / f r u i t borer Generallst external f o l l v o r e Generallst 31 31 external f o l l v o r e Salicaeae, Compositae Guttlferae 31 Noctuidae external f o l l v o r e Guttlferae 31 Noctuidae external f o l l v o r e Guttlferae 31 Geometrldae external f o l l v o r e Generallst Geometrldae external f o l l v o r e Generallst Sandberg, in prep.

Delta ramosula Delta stewarti Hyperetls amicarla Eupithecia miserulata Pleuroprucha lnsularia Platynota flavedana Sparganothls sulfureana Xenotemna pallorana Choristoneura parallela Unidentified sp.

Mode of feeding

Ref

Host range

Lycaenidae Noctuidae Noctuidae

Geometrldae external f o l l v o r e

Generallst Sandberg, in prep.

T o r t r l c l d a e leaf tyer T o r t r l c l d a e leaf tyer

Generallst Sandberg, in prep. Generallst Sandberg, in prep.

T o r t r i c i d a e l e a f tyer

Generallst Sandberg, in prep.

T o r t r l c l d a e leaf folder

Generallst Sandberg, in prep.

Graclllarlidae leaf folder

Sandberg, in prep.

Table IV. Effects of hypericin on Placynota flavedana i n the presence and absence of l i g h t (S. Sandberg, i n p r e p a r a t i o n ) 4a. Survivorship (%) of Platynota flavedana to second i n s t a r (n » 40 i n each treatment) Full light Filtered light 1

Control d i e t 0.03% hypericin d i e t

80.0 50.0

85.0 77.5

2 1

A G* test of independence y i e l d e d a value of .109 f o r the Interaction of hypericin and l i g h t regime, i n d i c a t i n g the t o x i c i t y of hypericin i s affected by the l i g h t regime

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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211

Resistance to phototoxins Many i n s e c t s may r e l y on p h y s i c a l f a c t o r s f o r p r o t e c t i o n from p h o t o t o x i n s ; i n these c a s e s , a l t h o u g h the i n s e c t i n g e s t s o r c o n t a c t s p h o t o t o x i n s and i s exposed t o l i g h t , the l i g h t f a i l s t o r e a c h the target s i t e of the molecule. I n mammals, d a r k - s k i n n e d i n d i v i d u a l s a r e r e l a t i v e l y more immune t o the e f f e c t s o f i n g e s t i o n o f o r c o n t a c t w i t h p h o t o t o x i n s (32). T h i s r e s i s t a n c e i s a t t r i b u t a b l e t o the d i f f e r e n t i a l concentrations of melanin. M e l a n i n a c t s as a p h o t o p r o t e c t i v e agent i n s e v e r a l ways. M e l a n i n absorbs b o t h UV and v i s i b l e l i g h t and a c t s as a n e u t r a l d e n s i t y f i l t e r ; m e l a n i n c o n t a i n i n g melanosomes s c a t t e r incoming r a d i a t i o n and a t t e n u a t e the l i g h t ; m e l a n i n can absorb r a d i a n t energy and d i s s i p a t e i t as heat; i t can a l s o , as a s t a b l e f r e e r a d i c a l , a c t as a " b i o l o g i c e l e c t r o n exchange polymer" (32). A l t h o u g h much o f the brown o r b l a c k c o l o r a t i o n o f i n s e c t c u t i c l e i s a t t r i b u t a b l e t o t a n n i n g ( i . e . , the protein-quinone c r o s s l i n k a g c o l o r a t i o n i n many s p e c i e l e a s t two s p e c i e s o f i n s e c t s a s s o c i a t e d w i t h p l a n t s c o n t a i n i n g p h o t o t o x i n s a r e prone t o m e l a n l c mutations. Melanic l a r v a e of P a p i l i o machaon (the O l d World s w a l l o w t a i l ) , an a s s o c i a t e o f p h o t o t o x i c U m b e l l i f e r a e , a r e known t o o c c u r (34). Manduca s e x t a , the tobacco hornworm ( L e p l d o p t e r a : S p h i n g i d a e ) , feeds on the f o l i a g e of S o l a n a c e a e , I n c l u d i n g L y c o p e r s l c o n e s c u l e n t u m , the tomato, w h i c h i s r e p o r t e d t o c o n t a i n the p h o t o t o x i c furanocoumarin bergapten (35). A mutant form a r i s e s on o c c a s i o n i n which the n o r m a l l y t r a n s p a r e n t c u t i c l e t u r n s b l a c k i n the u l t i m a t e l a r v a l i n s t a r due t o h o r m o n a l l y mediated pigment d e p o s i t i o n (36). These i n d i v i d u a l s can comprise up to 10% o f n a t u r a l p o p u l a t i o n s (G. Kennedy, p e r s o n a l communication 1986). The p h o t o t o x i c f u r a n o c o u m a r i n x a n t h o t o x i n was t o p i c a l l y a p p l i e d i n acetone a t the r a t e o f 50 micrograms/g body weight t o the d o r s a l a r e a o f the t h o r a x o f u l t i m a t e i n s t a r c a t e r p i l l a r s w i t h normal p i g m e n t a t i o n . T h i s treatment i n the presence o f UV l i g h t r e s u l t e d i n major i n j u r y t o t h e pupae. S p e c i f i c a l l y , p u p a l wings f a i l e d t o form and t o s c l e r o t i z e p r o p e r l y . Seventy p e r c e n t o f t h e t r e a t e d i n d i v i d u a l s f a i l e d t o pupate a t a l l o r m a n i f e s t e d c u t i c u l a r damage t o wings. That the damage was e s s e n t i a l l y l i m i t e d t o the d e v e l o p i n g wings i s c o n s i s t w i t h the i n t e r p r e t a t i o n t h a t m i t o t i c a l l y a c t i v e t i s s u e (such as d e v e l o p i n g wing i m a g l n a l d i s c s ) i s p a r t i c u l a r l y s u s c e p t i b l e t o the a n t i m i t o t i c e f f e c t s o f i r r a d i a t e d f u r a n o c o u m a r i n s . When b l a c k mutant hornworm l a r v a e were t r e a t e d i n an i d e n t i c a l f a s h i o n i n t h e u l t i m a t e i n s t a r , o n l y 30% f a i l e d t o pupate o r e x h i b i t e d wing d e f o r m i t i e s (Wiseman and Berenbaum, i n p r e p a r a t i o n ) . M e l a n i n , then, appears t o c o n f e r p r o t e c t i o n a g a i n s t the p h o t o a c t i v a t i o n o f f u r a n o c o u m a r i n s by UV l i g h t and such p r o t e c t i o n may account f o r the p e r s i s t e n t presence o f m e l a n i c i n d i v i d u a l s i n some i n s e c t p o p u l a t i o n s . H i g h l y r e f l e c t i v e s u r f a c e s may a l s o c o n f e r some p r o t e c t i o n a g a i n s t p h o t o t o x i n s (37). S e v e r a l s p e c i e s o f c h r y s o m e l i d b e e t l e s are f r e q u e n t a s s o c i a t e s o f t h e genus Hypericum; o f t h e s e , s p e c i e s i n the genus C h r y s o l l n a a r e c h a r a c t e r i s t i c a l l y m e t a l l i c b l u e - b l a c k i n c o l o r . T h e i r h i g h l y r e f l e c t i v e s u r f a c e may p r e v e n t v i s i b l e l i g h t from e n t e r i n g the body c a v i t y t o a c t i v a t e i n g e s t e d h y p e r i c i n c o n t a i n i n g p l a n t t i s s u e (38). I n g e n e r a l m e t a l l i c c o l o r s , w h i l e

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

LIGHT-ACTIVATED PESTICIDES

212

a b s o r b i n g i n c i d e n t r a d i a t i o n w e l l , absorb p o o r l y . The m e t a l l i c c u t i c l e o f t i g e r b e e t l e s r e f l e c t s u b s t a n t i a l amounts of shortwave r a d i a t i o n , r a n g i n g from 280 t o 580 nm (39). Biochemical

resistance to

phototoxins

B i o c h e m i c a l r e s i s t a n c e to p h o t o t o x i n s has been documented i n s e v e r a l insects associated with phototoxic plants. Biochemical resistance i n v o l v e s m e t a b o l i s m of a t o x i n such t h a t i t i s no l o n g e r t o x i c . One g e n e r a l b i o c h e m i c a l defense a g a i n s t p h o t o t o x i n s i s to i n t e r c e p t a p h o t o a c t i v e m o l e c u l e w i t h a n o t h e r m o l e c u l e to m a i n t a i n i t i n the o s t e n s i b l y n o n t o x i c ground s t a t e (38). L a r s o n 1986 suggested t h a t many i n s e c t s produce o r s e q u e s t e r c h e m i c a l s w i t h the a b i l i t y to p h y s i c a l l y "quench" e x c i t e d s t a t e s — t h a t i s , to remove energy from an e x c i t e d s t a t e "donor" m o l e c u l e w i t h o u t undergoing s t r u c t u r a l change. Beta c a r o t e n e and r e l a t e d c a r o t e n o i d s , which are e x c e l l e n t quenchers, ar integument of h e r b i v o r o u quenchers f o r those s p e c i e g phototoxi plants a d d i t i o n , due to i t s a b s o r p t i o n maxima (around 450 t o 550 nm), b e t a c a r o t e n e may d i r e c t l y quench the p h o t o t o x i c f u r a n o c o u m a r i n s , w h i c h show maximal f l u o r e s c e n c e In t h a t r e g i o n . N i t r o g e n - c o n t a i n i n g pigments such as the p t e r i n e s and the ommochromes may a l s o be i n v o l v e d i n oxygen quenching, inasmuch as s i m i l a r a l k a l o i d s possess t h i s p r o p e r t y (41). T o x i c oxygen s p e c i e s are a l s o s u b j e c t to p h y s i c a l and b i o c h e m i c a l d e t o x i c a t i o n i n i n s e c t s t h a t feed on p h o t o t o x i c p l a n t s . C e r t a i n i n s e c t c o n s t i t u e n t s o f c u t i c l e o r hemolymph a r e , o r r e s e m b l e , s t r u c t u r a l l y e f f i c i e n t quenchers of s i n g l e t oxygen. These i n c l u d e c a r o t e n o i d s , amines, and s u l f u r and oxygen d e r i v a t i v e s (38). F l a v o n o i d pigments can a c t as e f f i c i e n t s i n g l e t oxygen s c a v e n g e r s as w e l l . Q u e r c i t i n , a w i d e l y d i s t r i b u t e d p l a n t c o n s t i t u e n t , can suppress s i n g l e t - o x y g e n dependent r e a c t i o n s (42). G l y c o s i d e s o f q u e r c i t i n appear to be s e l e c t i v e l y sequestered from t h e i r f o o d p l a n t s by s w a l l o w t a i l b u t t e r f l i e s i n the t r i b e G r a p h i i n i and by one s p e c i e s i n the genus P a p i l i o (43). P h o t o t o x i c a l k a l o i d s (e.g., b e r b e r i n e ) are r e p o r t e d to occur i n the annonaceous h o s t s of these b u t t e r f l i e s (43) and the u m b e l l i f e r o u s h o s t p l a n t s of P a p i l i o machaon, the s p e c i e s s e q u e s t e r i n g q u e r c i t i n g l y c o s i d e s , are known to c o n t a i n f u r a n o c o u m a r i n s , s e v e r a l of w h i c h g e n e r a t e s i n g l e t oxygen i n the presence o f UV (44). Other s p e c i e s of s w a l l o w t a i l s , p a r t i c u l a r l y i n the T r o i d i n i and P a p i l i o n i n i , s e l e c t i v e l y s e q u e s t e r c a r o t e n o i d s ; o v e r a l l the c o n c e n t r a t i o n of c a r o t e n o i d s i n P a p i l i o n i d a e i s up t o an o r d e r of magnitude h i g h e r than c o n c e n t r a t i o n s i n o t h e r b u t t e r f l y f a m i l i e s ( T a b l e V). R o t h s c h i l d et a l . (45) suggested t h a t c a r o t e n o i d s e q u e s t r a t i o n may s e r v e to p r o t e c t t r o i d i n e s a s s o c i a t e d w i t h A r i s t o l o c h i a c e a e by p r e v e n t i n g f r e e - r a d i c a l o x i d a t i o n of the n i t r o p h e n a n t h r e n e a r i s t o l o c h i c a c i d s to p h e n o l i c s . In a d d i t i o n , c a r o t e n o i d s may s e r v e as s i n g l e t oxygen quenchers f o r the s e v e r a l c l a s s e s of p h o t o s e n s i t i z e r s ( i n c l u d i n g f u r a n o c o u m a r i n s , f u r o q u i n o l i n e a l k a l o i d s , furochromones, and b e n z y l i s o q u i n o l i n e a l k a l o i d s ) present i n the Rutaceae, p r i n c i p a l host f a m i l y f o r the m a j o r i t y o f p a p i l i o n i n e s (46).

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

14.

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Phototoxicity as a Defense Against Insects

Table V.

Family Lycaenidae Nymphalidae Pieridae Satyridae Papilionidae*

213

C a r o t e n o i d content o f b u t t e r f l i e s ( 4 0 )

T o t a l ug/g d r y weight 79.4 62.1 32.7 70.4 297

* C a l c u l a t e d from R o t h s c h i l d ( 4 5 ) , Valadon and Mummery (47)

S p e c i f i c b i o c h e m i c a l pathways f o r d e t o x i f i c a t i o n are known t o e x i s t i n some s p e c i e s o p l a n t s . I v i e and c o l l e a g u e m i x e d - f u n c t i o n oxidase-mediated d e t o x i f i c a t i o n o f furanocoumarins by the b l a c k s w a l l o w t a i l P a p i l i o p o l y x e n e s . A l t e r n a t e b i o c h e m i c a l d e t o x i f i c a t i o n systems may e x i s t as w e l l . D e p r e s s a r i a p a s t i n a c e l l a , the p a r s n i p webworm, i s an o e c o p h o r i d c a t e r p i l l a r t h a t feeds e x c l u s i v e l y on the w i l d p a r s n i p , P a s t i n a c a s a t i v a , w h i c h c o n t a i n s s e v e r a l p h o t o t o x i c furanocoumarins (50). T o x i c i t y o f f u r a n o c o u m a r i n s i s not enhanced by mixed f u n c t i o n o x i d a s e i n h i b i t o r s such as t h e m e t h y l e n e d i o x y p h e n y l - c o n t a i n i n g m y r i s t i c i n ( J . N i t a o , i n p r e p a r a t i o n ) ; t h i s o b s e r v a t i o n suggests t h a t an a l t e r n a t e r o u t e i s i n f o r c e . In f a c t , s u b s t a n t i a l amounts o f o r a l l y a d m i n i s t e r e d x a n t h o t o x i n ( a furanocoumarin found i n the p a r s n i p h o s t p l a n t ) a r e r e c o v e r a b l e i n t a c t i n the s i l k and s i l k g l a n d s o f t h e c a t e r p i l l a r , r a i s i n g the p o s s i b i l i t y t h a t the p a r s n i p webworm s e q u e s t e r s p l a n t d e r i v e d p h o t o t o x i n s f o r i t s own defense when ensconced i n l a r v a l webbing o r pupal cocoon s i l k ( J . N i t a o , i n p r e p a r a t i o n ) . Ecological variation To a g r e a t e x t e n t , e c o l o g i c a l f a c t o r s can i n f l u e n c e the e f f i c a c y o f p h o t o t o x i c i t y as a defense a g a i n s t i n s e c t s . On a v e r y s m a l l s c a l e , shade a v a i l a b i l i t y may determine the r e l a t i v e s u s c e p t i b i l i t y o f i n d i v i d u a l p l a n t s I n a p o p u l a t i o n t o i n s e c t s . F o r example, w i l d p a r s n i p s grown under c o n d i t i o n s o f 50 o r 70% ambient l i g h t show a s i g n i f i c a n t r e d u c t i o n i n furanocoumarin c o n t e n t o f t h e f o l i a g e (Berenbaum and Z a n g e r l 1986); inasmuch as furanocoumarins a r e t o x i c t o many i n s e c t s , r e d u c t i o n s i n the f o l i a r c o n c e n t r a t i o n o f these compounds may render p l a n t s i n shady s p o t s more v u l n e r a b l e t o h e r b i v o r y . A l t h o u g h no s p e c i f i c p h o t o t o x i n has been i d e n t i f i e d i n wheat, shade reduces r e s i s t a n c e o f hard r e d s p r i n g wheat t o the wheat stem s a w f l y Cephus c i n c t u s ; i n t h i s case, reduced p h o t o s y n t h e t i c e f f i c i e n c y may have reduced p l a n t v i g o r o r p r o d u c t i o n by t h e p l a n t o f o t h e r d e f e n s i v e c h e m i c a l s (51). S i n c e many a l l e l o c h e m i c a l s a r e induced by l i g h t , v a r i a t i o n i n l i g h t i n t e n s i t y can g r e a t l y a f f e c t t h e c h e m i c a l c o m p o s i t i o n o f above ground p l a n t p a r t s (Berenbaum 1987).

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Geographic v a r i a t i o n may a f f e c t the e f f i c a c y o f p h o t o t o x i c i t y as a p l a n t defense a g a i n s t i n s e c t s . G l o b a l v a r i a t i o n i n the i n c i d e n c e o f UV and v i s i b l e l i g h t i s s u b s t a n t i a l . T o x i c i t y o f a p h o t o t o x i n can be d i r e c t l y p r o p o r t i o n a l t o UV i n t e n s i t y (Berenbaum and Z a n g e r l 1987), so an e q u i v a l e n t c o n c e n t r a t i o n o f p h o t o t o x i n a t a h i g h e r l a t i t u d e , where i n c i d e n t UV i s a t t e n u a t e d , may have reduced t o x i c i t y . G l o b a l r a d i a t i o n i n t e n s i t y i s determined by a number o f f a c t o r s i n c l u d i n g s o l a r a n g l e , e l e v a t i o n above s e a l e v e l , a t m o s p h e r i c ozone c o n c e n t r a t i o n , atmospheric t u r b i d i t y , degree o f c l o u d c o v e r , and d i s t a n c e to t h e sun a t any p o i n t i n time (52). An i n c r e a s e i n a l t i t u d e from s e a l e v e l t o 4300 m corresponds t o an i n c r e a s e i n UV r a d i a t i o n o f 66% (53). L a t i t u d i n a l d i f f e r e n c e s a l s o a f f e c t UV i n t e n s i t i e s , l a r g e l y due t o g l o b a l d i f f e r e n c e s i n t h e d i s t r i b u t i o n o f atmospheric ozone c o n c e n t r a t i o n s ; g r e a t e r c o n c e n t r a t i o n s o f ozone a t h i g h l a t i t u d e s g r e a t l y reduce the i n t e n s i t y o f b i o l o g i c a l l y e f f e c t i v e UV r a d i a t i o n ( C a l d w e l l 1974). This sort of g l o b a l v a r i a t i o may account f o r t h e o b s e r v a t i o endogenous p h o t o t o x i n s appea region i n t e n s e s o l a r r a d i a t i o n i s a v a i l a b l e throughout most o f t h e year (e.g., i n t r o p i c a l o r a r i d d e s e r t ecosystems). Conclusions Many p l a n t f a m i l i e s have converged upon a common mechanism o f defense a g a i n s t h e r b i v o r o u s i n s e c t s , t h a t i s , t o e x p l o i t t h e abundant energy a v a i l a b l e i n s u n l i g h t t o p o t e n t i a t e endogenous secondary c h e m i c a l s . I t i s t h e r e f o r e h a r d l y s u r p r i s i n g t h a t , o v e r e v o l u t i o n a r y t i m e , h e r b i v o r o u s I n s e c t s have d e v e l o p e d v a r i o u s and sundry r e s i s t a n c e mechanisms t o these l i g h t - a c t i v a t e d defense compounds. These i n c l u d e b e h a v i o r a l , p h y s i c a l and b i o c h e m i c a l a d a p t a t i o n s t o reduce t h e e x t e n t o f exposure t o e i t h e r the t o x i n o r to p o t e n t i a t i n g w a v e l e n g t h s o f l i g h t , o r t o d i s m a n t l e and d i s a r m the t o x i n i t s e l f . W h i l e l i g h t - a c t i v a t e d p h y t o c h e m i c a l s may w e l l have p o t e n t i a l a p p l i c a t i o n s f o r c o n t r o l purposes i n a g r i c u l t u r a l entomology, these p h y t o c h e m i c a l s may be as prone t o c o u n t e r a d a p t a t i o n by i n s e c t s as a r e the more t r a d i t i o n a l s y n t h e t i c o r g a n i c c o n t r o l c h e m i c a l s — p e r h a p s more so, s i n c e t h e r e a l r e a d y e x i s t s a s u b s t a n t i a l group o f i n s e c t s preadapted t o f e e d i n g on p h o t o t o x i c p l a n t s . M o r e o v e r , t h e r e a r e e c o l o g i c a l c o n s t r a i n t s on the use o f p h o t o t o x i n s f o r widespread i n s e c t c o n t r o l . L o c a l v a r i a t i o n s i n l i g h t regime due t o such u n c o n t r o l l a b l e f a c t o r s as c l o u d c o v e r o r atmospheric t u r b i d i t y , o r t o such u n m o d i f i a b l e f a c t o r s as a l t i t u d e o r l a t i t u d e , may render a s t a n d a r d p h o t o t o x i n based c o n t r o l program a t best u n p r e d i c t a b l e . Acknowledgments I t h a n k E. H e i n i n g e r , R. L a r s o n , J . N e a l , J . N i t a o , and S. S a n d b e r g f o r comments on the m a n u s c r i p t and f o r a l l o w i n g me t o c i t e u n p u b l i s h e d d a t a . T h i s r e s e a r c h was supported by a N a t i o n a l Science Foundation P r e s i d e n t i a l Young I n v e s t i g a t o r Award (NSF BSR 8351407).

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BERENBAUM

Phototoxicity as a Defense Against Insects

215

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25. 26.

27. 28. 29.

Berenbaum, M.; N e a l , J.J. J . Chem. E c o l . 1986, 12, 809-812. Fraenkel, G.S. Science 1959, 129, 1466-1470. Berenbaum, M. Science 1978, 201, 532-534. Croteau, R.; Burbott, A.J.; Loomis, W.D. Phytochem. 1972, 11, 2937-2948. Arnason, T.; Swain, T.; Wat, C.K.; Graham, E.A.; P a r t i n g t o n , S.; Towsers, G.H.N. Biochem. System. E c o l . 1981, 9, 63-68. McLachlan, D.; Arnason, J.T.; P h i l o g e n e , B.J.R.; Champagne, D. Experientia 1982, 38, 1061-1062. Champagne, D.E.; Arnason, J.T.; P h i l o g e n e , B.J.R.; Morand, P.; Lam, J . J . Chem. Ecol., 1986, 12, 835-858. P h i l o g e n e , B.J.R.; Arnason, J.T.; Towers, G.H.N.; Campos, F.; Champagne, D.; McLachlan, D. J . Chem. Ecol. 1984, 10, 115-123. Arnason, T.; Towers, G.H.N.; P h i l o g e n e , B.J.R.; Lambert, J.D.H. Am. Chem. Soc. Symposium Ser 1983 208 140-151 Knox, J.P.; Dodge, A.D Kagan, J.; Szczepanski J. Chem. Ecol. 1986, 12, 899-914. Murray, R.H.; Mandez, J.; Brown, S. The Natural Coumarins; John Wiley and Sons, Ltd.: London. Muckensturm, B.; Duplay, P.C.R.; Simonis, M.T.; K i e n l e n , J.C. Biochem. System. Ecol. 1981, 9, 289-292. Yajima, T.; Kato, N.; Munakata, K. A g r i c . Biol. Chem. 1977, 41, 1263-1268. P h i l o g e n e , B.J.R.; Arnason, J.T.; D u v a l , F. Can. Ent. 1985, 117,1153-1157. Downum, K.R.; R o s e n t h a l , G.A.; Towers, G.H.N. Pest. Biochem. Physiol. 1984, 22, 104-109. Kagan, J . ; Chan, G. Experientla 1983, 39, 402-403. Waller, G.R.; Nowacki, E.K. A l k a l o i d Biology and Metabolism in Plants; Plenum Press: New York, 1978. Towers, G.H.N. Can. J. Bot. 1984, 62, 2900-11. Pate, D.W. Econ. Bot. 1983, 37, 396-405. Ohlsson, A.B.; Bjork, L.; Gatenbeck, S. Phytochem. 1983, 22, 2447-2450. Arakawa, O.; Hori, Y.; Ogata, R. Physiologia Plantarum 1985, 3, 64. Manuwoto, S.; S c r i b e r , J.M. Ag. E c o s y s t . Env., 1985,14,221236. H e l l e r , W.; Egin-Buehler, B.; Gardiner, S.; Knobloch, K-H.; Matern, U.; Ebel, J.; Hahlbrock, K. Plant Physiol., 1979, 64, 371-373. Hart, S.; Kogan, M.; Paxton, J. J . Chem. E c o l . 1983, 9, 657-672. Schultz, J.C. In V a r i a b l e Plants and Herbivores i n Natural and Managed Systems; Denno, R.F. and McClure, M.S., Eds.; Academic Press: New York, 1983; pp. 61-90. Lincoln, D.; Langenheim, J.H. Biochem. System. E c o l . 1978, 6, 21-32. E h r l i c h , P.; Raven, P. Evolution 1964, 18, 586-608. Robberecht, R.; Caldwell, M.M. Plant, Cell, and Env. 1983, 6, 477-485.

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30. Duran, N.; Song, P.-S.; Photochem. P h o t o b i o l . , 1986, 43, 677680. 31. Tietz, H.M. An Index to the Described L i f e Histories, E a r l y Stages and Hosts of the Macrolepidoptera of the Continental United States and Canada; A.C. A l l y n : Sarasota (FL), 1972. 32. Pathak, M.A.; Jimbow, K.; Szabo, G.; F i t z p a t r i c k , T.B. Photochem. Photobiol. Rev. 1974, 1, 211-239. 33. Chapman, R.F. The I n s e c t s — S t r u c t u r e and Function; E l s e v i e r : New York, 1971. 34. Gardiner, B.O.C. J . Res. Lep. 1976, 15, 184. 35. Mendez, J.; Brown, S.A. Can. J . Bot. 1971, 49, 2097-2100. 36. Safranek, L.; Riddiford, L.M. J. Insect Physiol. 1975, 21, 1931-1938. 37. Pathak, M.A.; F i t z p a t r i c k , T.B. In Sunlight and Man; F i t z p a t r i c k , T.B., Ed.; University of Tokyo Press: Tokyo, 1974; pp. 725-740. 38. Larson, R.A. J . Chem. E c o l . 1986, 12, 859-870. 39. Van Natto, C.; F r e i t a g 40. Czeczuga, B.; Biochem 41. Larson, R.A.; Marley, K.A. Phytochem. 1984, 23, 2351-2354. 42. Takahama, U.; Youngman, R.J.; E l s t n e r , E.F. Photobiochem. Photobiophys. 1984, 7, 175-181. 43. Wilson, A. Phytochem. 1986, 25, 1309-1313. 44. J o s h i , P.C.; Pathak, M.A. Biochem. Biophys. Res. Comm. 1983, 112, 638-646. 45. R o t h s c h i l d , M; Mummery, R.; Farrell, C. Bio. J. L i n n . Soc. 1986, 28, 359-372. 46. Feeny, P.; Rosenberry, L.; Carter, M. In Herbivorous Insects Host-seeking Behavior and Mechanisms; Ahmad, S., Ed.; Academic P r e s s : New York, 1983; pp. 27-76. 47. Valadon, L.R.G.; Mummery, R.S. Comp. Biochem. P h y s i o l . 1978, 61B, 359-372. 48. Bull, D.L.; I v i e , G.W.; B e i e r , R.C.; Pryor, N.W. J. Chem. Ecol., 1986, 12, 885-892. 49 I v i e , G.W.; Bull, D.L.; B e i e r , R.C.; Pryor, N.W. J . Chem. E c o l . 1986, 12, 871-884. 50. Berenbaum, M.; Zangerl., A.; N i t a o , J. Phytochem., 1984, 23, 1809-1810. 51. Holmes, N.D. Can. Ent. 1984, 116, 677-684. 52. Caldwell, M.M. In Photophysiology; Giese, A.C., Ed.; 1971; V o l . 6, 131-177. 53. Caldwell, M.M. Ecol. Monog., 1968, 38, 243-268. 54. Downum, K.R.; Rodriguez, E.; J . Chem. E c o l . , 1986, 12, 823-834. R E C E I V E D March10,1987

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 15

Biological Actions and Metabolic Transformations of Furanocoumarins G. Wayne Ivie Agricultural Research Service, U.S. Department of Agriculture, College Station, TX 77841

Furanocoumarins ar molecules that occu large number of plant families. Furanocoumarins have important uses in human medicine, are potent phototoxins to both man and domestic animals, are important host resistance mediators i n a number of plant species, and exhibit t o x i c i t y against a wide range of organisms. Furanocoumarin b i o l o g i c a l actions are expressed most potently upon activation by long wavelength u l t r a v i o l e t l i g h t , but these compounds also have l i g h t Independent actions--by mechanisms that are at present not understood. In mammals, birds, and insects, furanocoumarins are often rapidly metabolized and excreted, and i n insects, the rate of metabolism i s the major determinant of r e l a t i v e tolerance to these compounds in the d i e t . Metabolic mechanism i n animals include O-alkyl hydrolysis, hydrolysis of the pyrone ring, and oxidative opening of the furan ring, i n addition to other oxidative, reductive, and conjugative reactions.

Furanocoumarins o c c u r n a t u r a l l y as secondary m e t a b o l i t e s i n h i g h e r p l a n t s (J_). These compounds have been i s o l a t e d f r o m w e l l over a hundred p l a n t s p e c i e s r e p r e s e n t i n g a t l e a s t e i g h t f a m i l i e s , a l t h o u g h the U m b e l l i f e r a e and Rutaceae appear to have, In p a r t i c u l a r , l a r g e numbers of s p e c i e s t h a t c o n t a i n furanocoumarins ( 2 ) . S p e c i f i c furanocoumarin d e r i v a t i v e s g e n e r a l l y a r i s e i n n a t u r e from two c o n f i g u r a t i o n s of the b a s i c t r i c y c l i c r i n g s t r u c t u r e ( F i g u r e 1 ) . The number of d i s t i n c t f u r a n o c o u m a r i n s t r u c t u r e s p r e s e n t l y known from p l a n t s i s q u i t e l a r g e — w e l l over 200 d i f f e r e n t furanocoumarin s t r u c t u r e s have thus f a r been i d e n t i f i e d ( 3 ) . Most i n d i v i d u a l furanocoumarins are d i s t i n g u i s h e d by a l k o x y or a l k y l s u b s t i t u t i o n at e i t h e r of the two a v a i l a b l e a r o m a t i c p o s i t i o n s , a t the a v a i l a b l e carbons of the f u r a n r i n g o r , much l e s s p r e d o m i n a n t l y , This chapter not subject to U.S. copyright Published 1987 American Chemical Society

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F i g u r e 1. S t r u c t u r e s i n d i c a t i n g the r i n g f u s i o n o f l i n e a r furanocoumarins ( P s o r a l e n ) and a n g u l a r furanocoumarins (Isopsoralen).

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IVIE

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the pyrone r i n g . Many n a t u r a l l y - o c c u r r i n g furanocoumarins e x h i b i t s a t u r a t i o n of the f u r a n o l e f i n l c m o i e t y . M o n o s u b s t l t u t e d and d i s u b s t i t u t e d furanocoumarins are common, but t r i - or g r e a t e r s u b s t i t u t i o n i s r a r e . The pathways i n v o l v e d i n the b i o s y n t h e s i s of furanocoumarins have been t h o r o u g h l y s t u d i e d and are a t p r e s e n t q u i t e w e l l understood (4-6). Mode of B i o l o g i c a l A c t i o n s L i g h t Dependent A c t i o n s . Furanocoumarins are of i n t e r e s t from a g r i c u l t u r a l , m e d i c i n a l , p u b l i c h e a l t h , and e n v i r o n m e n t a l v i e w p o i n t s because they are h i g h l y p h o t o b i o l o g l c a l l y a c t i v e . Wavelengths i n the near u l t r a v i o l e t (320-360 nm) are the most e f f e c t i v e a c t i v a t i n g w a v e l e n g t h s , even though furanocoumarins absorb r e l a t i v e l y p o o r l y i n this region (7J. I t i s g e n e r a l l y a c c e p t e d t h a t the p h o t o b i o l o g i c a l a c t i o n s of furanocoumarins r e s u l t , i n t e r c a l a t i o n I n t o the doubl a c t i v a t i o n , they form c y c l o b u t a n pyrimidin Both the f u r a n and pyrone r i n g double bonds are p o t e n t i a l a l k y l a t i n g m o i e t i e s ; the l i n e a r furanocoumarins ( p s o r a l e n s ) are known t o form b o t h mono- and d i a d d u c t s ( c r o s s l i n k s ) , whereas the a n g u l a r c o n f i g u r a t i o n of the i s o p s o r a l e n s p e r m i t s o n l y monoadduction (8^). A l t h o u g h DNA p h o t o a l k y l a t i o n i s a w e l l - d e f i n e d m o l e c u l a r event a s s o c i a t e d w i t h furanocoumarin i n t e r a c t i o n s w i t h l i v i n g m a t t e r , r e c e n t s t u d i e s have produced evidence t h a t furanocoumarins b i n d w i t h s p e c i f i c , s a t u r a b l e , h i g h a f f i n i t y s i t e s on or i n mammalian c e l l s and t h a t such b i n d i n g i s to some e x t e n t i r r e v e r s i b l e upon UV exposure ( 9 ) . I t was proposed t h a t s p e c i f i c r e c e p t o r b i n d i n g phenomena as modes of a c t i o n , r a t h e r than s i m p l y the a l k y l a t i o n of DNA, might be more c o n s i s t e n t w i t h the known d i v e r s i t y of furanocoumarin b i o l o g i c a l a c t i o n s ( 9 ) . L i g h t - I n d e p e n d e n t A c t i o n s . A l t h o u g h furanocoumarins are known p r i m a r i l y f o r t h e i r l i g h t - c a t a l y z e d r e a c t i o n s , they n e v e r t h e l e s s have demonstrated b i o l o g i c a l a c t i v i t i e s i n the absence of a c t i v a t i n g r a d i a t i o n . Furanocoumarins are moderately t o x i c to l a b o r a t o r y mammals i n the dark, from both acute and subacute s t a n d p o i n t s ( 8 , 1 0 ) . Furanocoumarins are a l s o somewhat t o x i c to c e r t a i n i n s e c t s i n the d a r k , but such may r e s u l t from a n t i f e e d a n t a c t i v i t y more so than i n h e r e n t t o x i c i t y per se ( 1 1 ) . Some furanocoumarins are weakly mutagenic i n the absence of l i g h t , by thus f a r u n e x p l a i n e d mechanisms (12-16). C e r t a i n of the e n v i r o n m e n t a l e f f e c t s of furanocoumarins ( v i d e i n f r a ) are c l e a r l y l i g h t Independent b u t , a g a i n , the mechanisms i n v o l v e d are not known. R o l e o f Oxygen i n Furanocoumarin A c t i o n . In the p h o t o a d d u c t i o n of furanocoumarins w i t h DNA, t h e r e i s c l e a r l y no d i r e c t involvement of oxygen. However, the p h o t o g e n e r a t i o n of s i n g l e t oxygen by furanocoumarins has been w e l l documented, and such r e a c t i o n s may p o t e n t i a l l y be r e s p o n s i b l e f o r d i r e c t enzyme i n a c t i v a t i o n and membrane d i s r u p t i o n ( 1 7 ) . M o n o f u n c t i o n a l ( a n g u l a r ) furanocoumarins appear to be more e f f i c i e n t g e n e r a t o r s of s i n g l e t oxygen than a r e l i n e a r furanocoumarins ( 1 7 ) . They a l s o appear t o be more p h o t o c a r c i n o g e n i c than the b i f u n c t i o n a l ( l i n e a r ) f u r a n o c o u m a r i n s ,

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and i t has been suggested t h a t a c t i v a t e d oxygen may p l a y a major r o l e i n the p h o t o c a r c i n o g e n l c i t y of these compounds ( 2 ) . Other s t u d i e s , however, have shown t h a t s i n g l e t oxygen g e n e r a t i o n i s not s i g n i f i c a n t l y c o r r e l a t e d w i t h the g e n o t o x i c i t y of e i t h e r l i n e a r o r a n g u l a r f u r a n o c o u m a r i n s , u s i n g an e x c i s i o n r e p a i r d e f i c i e n t s t r a i n of IS. c o l i as the t e s t organism ( 1 8 ) . S i m i l a r l y , n e i t h e r s i n g l e t oxygen (19) n o r s u p e r o x i d e r a d i c a l (20) f o r m a t i o n c o u l d be c o r r e l a t e d w i t h s k i n p h o t o s e n s i t i z i n g a c t i v i t y amongst a c o n s i d e r a b l e a r r a y of l i n e a r and a n g u l a r furanocoumarins. Structure-Activity Considerations A v a i l a b l e d a t a on the s k i n p h o t o s e n s i t i z i n g a c t i v i t y of furanocoumarins i n d i c a t e t h a t , g e n e r a l l y , l i n e a r furanocoumarins a r e more b i o l o g i c a l l y a c t i v e than the a n g u l a r analogs ( 8 ) . Recent s t u d i e s have, however, p r o v i d e d d a t a to suggest t h a t under n a t u r a l c o n d i t i o n s of m u I t i w a v e l e n g t h l i g h t a c t i v a t i o n th i n h e r e n t biological activities o furanocoumarins may not g e n e r a l l y h e l d t h a t p h o t o s e n s i t i z i n g a c t i v i t y decreases w i t h i n c r e a s i n g c h e m i c a l c o m p l e x i t y of the a l k y l or a l k o x y s u b s t l t u e n t ( 8 ) , but some s t u d i e s have shown t h a t some of the more c h e m i c a l l y complex (and more p o l a r ) furanocoumarins are h i g h l y p h o t o t o x i c when the s k i n , a b a r r i e r t o a b s o r p t i o n , i s bypassed ( 2 2 ) . A r y l h y d r o x y l a t e d f u r a n o c o u m a r i n s , s e v e r a l of which occur i n n a t u r e , a r e i n a c t i v e as s k i n p h o t o s e n s i t i z e r s ( 8 ) . S t r u c t u r e - a c t i v i t y c o r r e l a t i o n s f o r furanocoumarins w i t h r e s p e c t t o b i o l o g i c a l a c t i o n s o t h e r than s k i n p h o t o s e n s i t i z a t i o n are e i t h e r i n c o m p l e t e or lacking. T o x i c o l o g i c a l and Other B i o c h e m i c a l E f f e c t s Because furanocoumarins are p o t e n t DNA p h o t o a l k y l a t l n g a g e n t s , I t i s not s u r p r i s i n g t h a t they show c o n s i d e r a b l e p h o t o t o x i c i t y toward a wide v a r i e t y of l i f e forms. Upon l i g h t a c t i v a t i o n , furanocoumarins are p o w e r f u l a n t i m i c r o b i a l agents ( 8 , 2 3 ) , nematocides ( 2 4 ) , i n s e c t i c i d e s ( 2 5 , 2 6 ) , o v i c i d e s ( 2 7 ) , and p o w e r f u l s k i n p h o t o s e n s i t i z e r s a g a i n s t man (8) and a n i m a l s ( 2 8 ) . They are a l s o possibly h e r b i c i d a l (29). Furanocoumarins are m o l l u s c i c i d a l (30,31) and p i s c i c i d a l ( 8 ) , but the r o l e of l i g h t i n these e f f e c t s i s u n c l e a r . C o n s i d e r i n g the known m o l e c u l a r events a s s o c i a t e d w i t h the l i g h t - s e n s i t i z e d i n t e r a c t i o n of furanocoumarins w i t h l i v i n g m a t t e r , i t seems almost c e r t a i n t h a t furanocoumarins I n the presence of a c t i v a t i n g l i g h t would be p o t e n t i a l l y p h o t o t o x i c to almost any form of l i f e . In a d d i t i o n t o acute p h o t o t o x i c o l o g i c a l e f f e c t s , furanocoumarins are h i g h l y photomutagenic and a r e , i n f a c t , mammalian p h o t o c a r c i n o g e n s ( 3 2 - 3 4 ) . Furanocoumarins are known to have v a r i o u s l i g h t independent e f f e c t s on some mammalian and I n s e c t enzyme systems. The l i n e a r f u r a n o c o u m a r i n , x a n t h o t o x i n (8-methoxypsoralen) induced mouse and/or r a t a r y l hydrocarbon h y d r o x l y a s e ( 3 5 , 3 6 ) , e t h y l morphone N-demethylase ( 3 6 ) , p - n i t r o a n i s o l e - O - d e m e t h y l a s e (35) and 7-ethoxycoumarin-O-deethylase ( 3 7 ) ; i t a l s o shortened h e x o b a r b i t a l s l e e p i n g time ( 3 8 ) , and i t may i n c r e a s e l e v e l s of cytochrome P-450

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(36) a l t h o u g h t h e r e are c o n f l i c t i n g data ( 3 7 ) . X a n t h o t o x i n d i d not induce a n i l i n e h y d r o x y l a s e ( 3 6 ) . I n t e r e s t i n g l y , p s o r a l e n , i s o p s o r a l e n , and 4 , 5 * , 8 - t r i m e t h y l p s o r a l e n a t e q u i v a l e n t doses f a i l e d to e x h i b i t any enzyme i n d u c t i o n p o t e n t i a l (35,36). I n the f a l l armyworm (Spodoptera f r u g i p e r d a ) d i e t a r y x a n t h o t o x i n induced midgut g l u t a t h i o n e ^ - t r a n s f e r a s e and h e p t a c h l o r e p o x l d a s e , i n c r e a s e d cytochrome P-450 c o n t e n t , b u t i n h i b i t e d a l d r i n e p o x i d a s e , b i p h e n y l - 4 - h y d r o x y l a s e , and p - c h l o r o - N - m e t h y l a n i l i n e N-demethylase (39). Environmental I n t e r a c t i o n s On the b a s i s of r e s e a r c h o b s e r v a t i o n s t o date, the most p l a u s i b l e e x p l a n a t i o n f o r the o c c u r r e n c e o f furanocoumarins i n h i g h e r p l a n t s i s t h a t these compounds e v o l v e d as defense chemicals a g a i n s t p l a n t pathogens and h e r b i v o r e s , and as a l l e l o p a t h l c agents t o enhance c o m p e t i t i v e n e s s amongst i n h i b i t seed g e r m i n a t i o are almost c e r t a i n l y l i g h t - i n d e p e n d e n expressio i n the s o i l environment. The avoidance of a u t o t o x i c i t y i s a p p a r e n t l y a c c o m p l i s h e d , a t l e a s t I n p a r t , through l o c a l i z a t i o n and/or s e q u e s t r a t i o n phenomena ( 4 0 ) . Furanocoumarins are w e l l e s t a b l i s h e d as p h y t o a l e x l n s . The i n f e c t i o n of both c e l e r y and p a r s n i p w i t h c e r t a i n p a t h o g e n i c organisms r e s u l t s i n g r e a t l y enhanced b i o s y n t h e s i s and a c c u m u l a t i o n of these compounds ( 4 2 ) ; enhanced b i o s y n t h e s i s of furanocoumarins i s a l s o e l i c i t e d by a number of o t h e r s t i m u l i as w e l l ( 4 3 ) . The a n t i f e e d a n t p r o p e r t i e s o f furanocoumarins are w e l l e s t a b l i s h e d f o r a number of i n s e c t s , i n c l u d i n g s e v e r a l Spodoptera s p e c i e s (25,44-46), Mythimna u n l p u n c t a t a ( 4 7 ) , and L e p t i n o t a r s a d e c e m l i n e a t a ( 4 7 ) . C o n v e r s e l y , i n s e c t s t h a t are adapted t o feed on furanocoumarin-containing p l a n t s may p e r c e i v e these compounds as o v i p o s i t i o n s t i m u l a n t s ( 4 8 ) , and i n a t l e a s t one i n s e c t , the b l a c k s w a l l o w t a i l b u t t e r f l y , ( P a p i l i o p o l y x e n e s ) , d i e t a r y furanocoumarins a c t u a l l y enhance c a t e r p i l l a r growth r a t e and w e i g h t g a i n , perhaps by a c t i n g as f e e d i n g s t i m u l a n t s ( 4 9 ) . Furanocoumarins i n p l a n t s pose c l e a r and documented hazards t o g r a z i n g mammals. P h o t o s e n s i t l z a t l o n of domestic c a t t l e , sheep, and p o u l t r y by d i e t a r y furanocoumarins has been documented ( 2 8 ) , and there are numerous i n s t a n c e s of p h o t o s e n s i t l z a t l o n i n man a s s o c i a t e d w i t h dermal p l a n t exposures ( 8 ) . The impact of furanocoumarins on mammalian and a v i a n w i l d l i f e s p e c i e s i s e s s e n t i a l l y unknown, but i t i s l i k e l y t h a t most w i l d l i f e s p e c i e s have adapted through e v o l u t i o n a r y p r e s s u r e s t o a v o i d such p l a n t s . E v i d e n c e has r e c e n t l y been o b t a i n e d t h a t furanocoumarins are p r o b a b l y a n t i f e e d a n t s toward at l e a s t one mammalian h e r b i v o r e , the h y r a x ( P r o c a v l a c a p e n s l s ) (50). M e d i c i n a l Uses The v a r i o u s p h o t o b l o l o g l c a l a c t i o n s e x h i b i t e d by furanocoumarins a r e such t h a t these compounds have an astounding range of a c t u a l and p o t e n t i a l uses i n human m e d i c i n e . P l a n t p r e p a r a t i o n s t h a t c o n t a i n f u r a n o c o u m a r i n s , p l u s s u n l i g h t , have been used f o r thousands o f y e a r s i n the treatment o f v i t i l i g o ( 8 ) , and x a n t h o t o x i n p l u s UV

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l i g h t (PUVA t h e r a p y ) i s now the treatment of c h o i c e f o r severe p s o r i a s i s (32,33), Other human d i s o r d e r s f o r which furanocoumarins ( u s u a l l y x a n t h o t o x i n ) p l u s l i g h t have shown p o t e n t i a l l y b e n e f i c i a l e f f e c t s I n c l u d e mycosis f u n g o i d e s ( 5 1 ) , scleromyxoedma ( 5 2 ) , u r t i c a r i a pigmentosa ( 5 3 , 5 4 ) , f o l l i c u l a r mucinosis ( 5 5 ) , p a r a p s o r i a s i s ( 5 6 ) , p a l m o p l a n t a r p u s t u l o s i s ( 5 7 ) , lymphomatold p a p u l o s i s ( 5 8 ) , l i c h e n planus ( 5 9 ) , a t o p i c d e r m a t i t i s ( 6 0 ) , c e r t a i n p a r a s i t i c f u n g i ( 6 1 ) , and even herpes s i m p l e x ( 6 2 ) , a l o p e c i a a r e a t a (63,64), and leukemic cutaneous T c e l l lymphoma ( 6 5 ) . Metabolic

Transformations

Mammals. The e x t e n s i v e I n t e r e s t i n l i n e a r furanocoumarins as m e d i c i n a l agents and t h e i r impact as l i v e s t o c k and p o u l t r y p h o t o t o x i n s has l e d t o c o n s i d e r a b l e s c i e n t i f i c e f f o r t s aimed a t e l u c i d a t i n g the k i n e t i c s , b i o t r a n s f o r m a t i o n and d i s p o s i t i o n of these c h e m i c a l s i n mammalian s p e c i e s have been t a r g e t e d on x a n t h o t o x i n of c h o i c e i n most m e d i c i n a a p p l i c a t i o n s , perhap commonly-occurring furanocoumarin i n n a t u r e , and i s h i g h l y p h o t o b i o l o g i c a l l y a c t i v e . However, l i m i t e d b i o l o g i c a l t r a n s f o r m a t i o n s t u d i e s have a l s o been done w i t h bergapten ( 5 - m e t h o x y p s o r a l e n ) , and w i t h 4 , 5 , 8 - t r i m e t h y l p s o r a l e n w h i c h i s c l i n i c a l l y u s e f u l i n the treatment o f v i t i l i g o . The I n v i v o and i n v i t r o metabolism of x a n t h o t o x i n i n l a b o r a t o r y r a t s and mice has been the s u b j e c t of s e v e r a l s t u d i e s (66-70). I n r o d e n t s , x a n t h o t o x i n i s m e t a b o l i z e d by 1) O-demethylation; 2) a r y l h y d r o x y l a t i o n a t p o s i t i o n 5; 3) o x i d a t i o n of t h e 5,8-dihydroquinone t o the qulnone; 4) h y d r o l y s i s of the pyrone r i n g ; 5) o x i d a t i v e opening of the f u r a n r i n g ; and 6) s u l f a t e and g l u c u r o n l d e c o n j u g a t i o n ( F i g u r e 2 ) . I n v i t r o s t u d i e s have demonstrated t h a t x a n t h o t o x i n metabolism I n r a t s i s induced by p h e n o b a r b i t a l and by B-naphthoflavone ( 7 0 ) . I n r a t s , t h e r e i s some i n d i c a t i o n t h a t cleavage of the a r o m a t i c r i n g o f x a n t h o t o x i n o c c u r s to a very l i m i t e d extent (69). As i n d i c a t e d i n F i g u r e 2, x a n t h o t o x i n metabolism I n the dog ( 7 1 ) , i n the goat ( 7 2 ) , and i n man (67,73-75) f o l l o w s a t l e a s t some of t h e same pathways. I n t h e goat, a n o v e l m e t a b o l i t e r e s u l t s from s a t u r a t i o n of the pyrone r i n g p r i o r o r subsequent t o pyrone r i n g h y d r o l y s i s . A l t h o u g h not e s t a b l i s h e d e x p e r i m e n t a l l y , t h i s m e t a b o l i t e may a r i s e through r e d u c t i v e mechanisms p r e s e n t i n t h e rumen p r i o r t o a b s o r p t i o n ( 7 2 ) . D e f i n i t i v e m e t a b o l i c f a t e s t u d i e s have not been undertaken w i t h b e r g a p t e n , but a s i n g l e study o f l i m i t e d scope has p r o v i d e d good e v i d e n c e t h a t , i n man, t h e pyrone r i n g of bergapten i s h y d r o l y z e d and s u b s e q u e n t l y c o n j u g a t e d w i t h g l u c u r o n i c a c i d o r s u l f a t e p r i o r t o e x c r e t i o n i n the urine (76). S t u d i e s w i t h 4 , 5 ' , 8 - t r i m e t h y l p s o r a l e n i n mouse and man have shown t h a t the 5 - m e t h y l group i s h y d r o x y l a t e d , then o x i d i z e d t o a 5'-carboxy d e r i v a t i v e ( F i g u r e 3) (77,78). Products of f u r a n o r pyrone r i n g cleavage r e a c t i o n s were not d e t e c t e d . f

1

B i r d s . There a r e no p u b l i s h e d data on the f a t e of furanocoumarins i n any a v i a n s p e c i e s , but s t u d i e s i n p r o g r e s s i n our l a b o r a t o r i e s have shown t h a t x a n t h o t o x i n i s e x t e n s i v e l y m e t a b o l i z e d by l a y i n g

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

F i g u r e 2. I d e n t i f i e d m e t a b o l i c pathways f o r x a n t h o t o x i n (8-methoxypsoralen) i n r o d e n t s , humans, dogs, g o a t s , c h i c k e n s , and i n s e c t s . B r a c k e t e d compounds have not been i s o l a t e d , but a r e p o s s i b l e i n t e r m e d i a t e s i n pathways l e a d i n g t o the i d e n t i f i e d metabolites.

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hens ( P a n g i l i n a n , N. C.; I v i e , G. W.; u n p u b l i s h e d d a t a ) . The h y d r o l y s i s of t h e O-methyl group o f x a n t h o t o x i n ( t o x a n t h o t o x o l ) o c c u r s t o a t l e a s t a l i m i t e d e x t e n t i n the hen, and p r e l i m i n a r y i n d i c a t i o n s a r e t h a t the major m e t a b o l i t e s a r i s e through f u r a n and/or pyrone r i n g cleavage r e a c t i o n s . I n s e c t s . A number of s t u d i e s have i n v e s t i g a t e d the d i s p o s i t i o n o f furanocoumarins i n i n s e c t s , w i t h the primary emphasis on e s t a b l i s h i n g how f u r a n o c o u m a r i n - t o l e r a n t s p e c i e s a v o i d p h o t o t o x i c i t y . Under c o n d i t i o n s of l a b o r a t o r y f e e d i n g of xanthotoxin t o aphids (Aphis h e r a c l e l l a o r C a v a r l e l l a pastinacae) c o n t i n u o u s l y exposed t o UV l i g h t , no p h o t o t o x i c e f f e c t was seen. No x a n t h o t o x i n m e t a b o l i t e s per se were d e t e c t e d i n e x t r a c t s of t r e a t e d a p h i d s , b u t x a n t h o t o x i n c o u l d be l i b e r a t e d by a c i d h y d r o l y s i s procedures ( 2 9 ) . I t may be t h a t aphids d e t o x i f y x a n t h o t o x i n by h y d r o l y s i s of the l a c t o n e f o l l o w e d by c o n j u g a t i o n (perhaps as a glycoside). Acid hydrolysi then would l i k e l y l a c t o n i z The l a r v a l form of a l e a f mining d i p t e r a n , Phytomyza s p o n d y l l l , feeds on p l a n t s r i c h i n furanocoumarins and has been shown t o r a p i d l y d e t o x i f y xanthotoxin to non-photoactive m e t a b o l i t e s , a l t h o u g h the c h e m i c a l n a t u r e of these p r o d u c t s has not been i n v e s t i g a t e d (79). C a t e r p i l l a r s of the b l a c k s w a l l o w t a i l b u t t e r f l y , P a p i l i o p o l y x e n e s , a r e w e l l adapted t o feed on l i n e a r furanocoumarin rich h o s t p l a n t s , and i t i s now known t h a t t h i s i n s e c t r a p i d l y d e t o x i f i e s l i n e a r furanocoumarins ( p s o r a l e n , x a n t h o t o x i n ) as the mechanism o f t o x i c i t y avoidance (80,81^). O x i d a t i v e cleavage of the f u r a n r i n g i s the major r o u t e o f d e t o x i f i c a t i o n by P. polyxenes ( F i g u r e 2 ) ; O - d e m e t h y l a t i o n , pyrone r i n g h y d r o l y s i s , o r o t h e r pathways a r e e i t h e r n o n - e x i s t e n t o r minor. Larvae of the furanocoumarin s e n s i t i v e f a l l arrayworm (Spodoptera f r u g i p e r d a ) m e t a b o l i z e l i n e a r furanocoumarins by i d e n t i c a l pathways, y e t a t such a slow r a t e t h a t t o x i c i t y ensues (80,81). polyxenes appears t o be r e l a t i v e l y l e s s t o l e r a n t t o a n g u l a r furanocoumarins (82) and, i n d e e d , an a n g u l a r furanocoumarin ( i s o p s o r a l e n ) was m e t a b o l i z e d a t a slower r a t e than was p s o r a l e n (83). T h i s o b s e r v a t i o n may a t l e a s t p a r t l y e x p l a i n why ]>. polyxenes g e n e r a l l y a v o i d s p l a n t s t h a t c o n t a i n a p p r e c i a b l e l e v e l s of the a n g u l a r compounds. The m e t a b o l i c d e t o x i f i c a t i o n of furanocoumarins i n l e p l d o p t e r a n l a r v a e r e s u l t s , at l e a s t I n p a r t , from the a c t i o n s o f microsomal o x i d a s e s . T h i s c o n c l u s i o n i s based on d i r e c t s t u d i e s w i t h x a n t h o t o x i n and midgut or body microsomes from P. polyxenes and S. f r u g i p e r d a ( 8 4 ) , and upon o b s e r v a t i o n s t h a t the t o x i c i t y ( i n the d a r k ) o f x a n t h o t o x i n t o the corn earworm, H e l l o t h l s z e a , i s enhanced by m y r i s t i c i n and p i p e r o n y l b u t o x i d e , potent methylenedioxyphenyl I n h i b i t o r s o f microsomal o x i d a s e enzymes (11)* Metabolism

Versus E x p r e s s i o n o f B i o l o g i c a l E f f e c t s

Given the f a c t t h a t furanocoumarins r e a d i l y p h o t o a l k y l a t e DNA, these compounds can be c o n s i d e r e d as n o n s p e c i f i c p h o t o s e n s i t i z e r s capable of i n t e r a c t i n g w i t h e s s e n t i a l l y any form of l i f e under a p p r o p r i a t e l i g h t a c t i v a t i o n s c e n a r i o s . I n s i n g l e c e l l e d organisms, such

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i n t e r a c t i o n s may l e a d t o c e l l u l a r d e a t h , the i n h i b i t i o n of c e l l u l a r d i v i s i o n and thus m u l t i p l i c a t i o n o r , a t a minimum, mutagenic e f f e c t s . I n m u l t i c e l l u l a r organisms, t h e p h o t o s e n s i t i z e d e f f e c t s are expected t o be l i m i t e d t o dermal and subdermal t i s s u e s , i . e . , l i m i t e d by the degree o f l i g h t p e n e t r a t i o n . I n mammals, p a r t i c u l a r l y , such I n t e r a c t i o n s may be t o x i c o l o g i c a l i n t h a t s e v e r e t i s s u e damage occurs (erythema, s k i n b l i s t e r i n g , c a r c i n o g e n i c i t y ) o r pharmacological i n that f u r a n o c o u m a r i n - l i g h t - t i s s u e i n t e r a c t i o n s result i n desired medicinal effects ( v i t i l i g o , p s o r i a s i s ) . The r a t e of furanocoumarin metabolism by any organism almost c e r t a i n l y governs the s e v e r i t y and d u r a t i o n of the p h o t o b i o l o g i c a l a c t i o n s a s s o c i a t e d w i t h these compounds. T h i s c o n c l u s i o n seems j u s t i f i e d i n t h a t e s s e n t i a l l y any l i k e l y m e t a b o l i c t r a n s f o r m a t i o n can be expected t o r e s u l t i n s i g n i f i c a n t o r t o t a l d i m i n u t i o n o f p h o t o r e a c t i v i t y and/or an i n c r e a s e d tendency toward r a p i d e x c r e t i o n . A r y l h y d r o x y l a t l o n o r O - a l k y l h y d r o l y s i s r e a c t i o n s render furanocoumarins p h o t o b i o l o g i c a l l i n a c t i v (8) and structural grounds, f u r a n o r pyron ( a l t h o u g h not y e t e s t a b l i s h e metabolites. C e r t a i n of the p o t e n t i a l i n t e r m e d i a t e s i n f u r a n o c o u m a r i n metabolism might, i n f a c t , r e t a i n p h o t o b i o l o g i c a l a c t i v i t y ( i . e . , t h e 4 , 5 - e p o x i d e , the quinone, and t h e 3,4-dihydro d e r i v a t i v e s , F i g u r e 2 ) . However, such compounds would be r a p i d l y subjected to a d d i t i o n a l degradation r e a c t i o n s . With 4 , 5 , 8 - t r i m e t h y l p s o r a l e n , mammalian metabolism a p p a r e n t l y i n v o l v e s methyl group o x i d a t i o n t o a g r e a t e r e x t e n t than r i n g c l e a v a g e ( i f indeed r i n g c l e a v a g e r e a c t i o n s o c c u r a t a l l ) , b u t the major metabolite (5'-carboxy dimethylpsoralen) i s p h o t o b i o l o g i c a l l y i n a c t i v e (77) and r e a d i l y e x c r e t e d . Reduced b i o l o g i c a l a c t i v i t y o f furanocoumarin m e t a b o l i t e s has a l s o been I n d i c a t e d by an observed r e d u c t i o n i n photomutagenic a c t i v i t y o f x a n t h o t o x i n a f t e r i n c u b a t i o n w i t h r a t l i v e r mixed f u n c t i o n oxidase enzymes ( 8 5 ) . The metabolism of furanocoumarins by h i g h e r organisms appears to be, almost u n i v e r s a l l y , q u i t e r a p i d . I n o r a l l y - d o s e d rodents and I n man, peak plasma l e v e l s of absorbed x a n t h o t o x i n u s u a l l y o c c u r w i t h i n 1-2 h o u r s , f o l l o w e d by r a p i d d e p l e t i o n (67,74,75). M e t a b o l i t e s a r e q u i c k l y , and p r i m a r i l y , e l i m i n a t e d i n the u r i n e (68,74,75). S i m i l a r l y , r a p i d r a t e s o f m e t a b o l i c d e t o x i f i c a t i o n and e x c r e t i o n a r e seen i n dogs ( 7 1 ) , t h e goat ( 7 2 ) , and i n b i r d s ( P a n g i l i n a n , N. C ; I v i e , G. W.; u n p u b l i s h e d d a t a ) . Even i n I n s e c t s not adapted t o d i e t a r y furanocoumarins (S. f r u g i p e r d a ) , m e t a b o l i s m and e x c r e t i o n a r e q u i t e r a p i d , a l t h o u g h f a r l e s s so than f o r t h e f u r a n o c o u m a r i n t o l e r a n t P. p o l y x e n e s . S i x hours a f t e r o r a l treatment o f S. f r u g i p e r d a w i t h x a n t h o t o x i n , o n l y about 6% of t h e a d m i n i s t e r e d dose remains u n e x c r e t e d as the parent compound ( 8 1 ) . A l t h o u g h furanocoumarins a r e i n g e n e r a l much more b i o l o g i c a l l y a c t i v e i n t h e presence of l o n g w a v e l e n g t h UV l i g h t , these compounds do have demonstrable l i g h t - i n d e p e n d e n t a c t i o n s ( v i d e s u p r a ) . It i s , i n g e n e r a l , p o o r l y known t o what extent b i o t r a n s f o r m a t i o n s might a f f e c t such a c t i o n s , a l t h o u g h i t i s p r o b a b l y t r u e t h a t u l t i m a t e m e t a b o l i s m would u s u a l l y r e s u l t I n d e r i v a t i v e s of decreased b i o l o g i c a l a c t i v i t y (see F i g u r e 2 ) . However, r a t l i v e r enzymes, i n v i t r o , a p p a r e n t l y m e t a b o l i z e both x a n t h o t o x i n and 4,5',8-trimethyl=» p s o r a l e n t o mutagenic d e r i v a t i v e s ( 1 4 ) . A l s o , some s y n t h e t i c a n g u l a r furanocoumarins ( w i t h h y d r o p h i l l c s u b s t i t u e n t s a t the 4 1

f

1

f

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p o s i t i o n of the f u r a n r i n g ) a r e mutagenic i n the dark, but o n l y a f t e r microsomal a c t i v a t i o n ( 8 7 ) . The nature of the mutagenic m e t a b o l i t e s of such compounds i s unknown. That m e t h y l e n e d i o x y p h e n y l compounds s y n e r g i z e the t o x i c i t y of x a n t h o t o x i n t o H e l i o t h i s i n the dark (11) i m p l i e s t h a t the a c t i o n s of mixed f u n c t i o n o x i d a s e enzymes i n t h i s i n s e c t are p r i m a r i l y of a d e t o x i f i c a t i o n n a t u r e . The e f f e c t s of m e t a b o l i c a l t e r a t i o n s of p o t e n t i a l l y b i o l o g i c a l l y a c t i v e s u b s t i t u e n t m o i e t i e s of furanocoumarins i s a l s o p o o r l y u n d e r s t o o d . I t i s known t h a t the s y n t h e t i c furanocoumarin d e r i v a t i v e , p s o r a l e n g l y c i d y l e t h e r , Is a potent l i g h t independent mutagen, but t h a t the a c t i o n of epoxide h y d r o l a s e s reduces m u t a g e n i c i t y , c l e a r l y through h y d r o l y s i s of the epoxide moiety ( 8 6 ) . Conclusions Furanocoumarins have perhaps the w i d e s t documented spectrum of biological activities o Because of t h e i r major l i g h t - c a t a l y z e l i v i n g matter, p r e s e n t l y , p o t e n t i a l l y e x e r t p h o t o b i o c h e m i c a l i n f l u e n c e s on e s s e n t i a l l y any l i f e form. Furanocoumarins a l s o have s i g n i f i c a n t l i g h t - i n d e p e n d e n t a c t i o n s , by mechanisms t h a t a r e , a t p r e s e n t , e s s e n t i a l l y u n s t u d i e d . S t u d i e s of the b i o c h e m i c a l f a t e of furanocoumarins i n a number of v e r t e b r a t e and I n v e r t e b r a t e s p e c i e s have p r o v i d e d d a t a of c o n s i d e r a b l e v a l u e In e s t a b l i s h i n g how these c h e m i c a l s i n t e r a c t w i t h v a r i o u s l i f e forms and i n e x p l a i n i n g the r e l a t i v e p h o t o s e n s i t i v i t y of d i f f e r e n t s p e c i e s t o f u r a n o c o u m a r i n s . A d d i t i o n a l m e c h a n i s t i c and f a t e s t u d i e s a r e , however, c l e a r l y needed t o assess the p o t e n t i a l r o l e of non D N A - a l k y l a t i o n m o d e s - o f - a c t i o n ( i . e . , r e c e p t o r b i n d i n g ) on both the l i g h t - d e p e n d e n t and l i g h t - i n d e p e n d e n t a c t i o n s of these t o x i c o l o g l c a l l y , p h a r m a c o l o g i c a l l y , a g r i c u l t u r a l l y , and e n v i r o n m e n t a l l y s i g n i f i c a n t compounds. Literature 1. 2. 3. 4. 5. 6.

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Sharp, D. E.; Mays, D. C.; Rogers, S. L.; Guiler, R. C.; Hecht, S.; Gerber, N. Proc. West. Pharmacol. Soc. 1984, 27, 255-258. K o l i s , S.; Williams, T.; Postma, E.; Sasso, G.; Confalone, P.; Schwartz, M. Drug Metab. Dispos. 1979, 7, 220-225. Ivie, G. W.; Beier, R. C.; B u l l , D. L.; O e r t l i , E. H. Am. J . Vet. Res. 1986, 47, 799-803. Ehrsson, H.; Eksborg, S.; Wallin, I. Eur. J . Drug Metab. Pharmacokinet. 1978, 2, 125-128. Busch, U.; Schmld, J . ; Koss, F. W.; Zipp, H.; Zimmer, A. Arch. Dermatol. Res. 1978, 262, 255-265. Schmid, J . ; Prox, A.; Reuter, A.; Zipp, H.; Koss, F. W. Eur. J. Drug Metab. Pharmacokinet. 1980, 5, 81-92. Stolk, L. M. L.; Westerhof, W.; Cormane, R. H.; Van Zwieten, P. A. Br. J . Dermatol. 1981, 105, 415-420. Mandula, B. B.; Pathak, M. A.; Dudek, G. Science 1976, 193, 1131-1134. Mandula, B. B.; Pathak 127-132. Ashwood-Smith, M. ; Ring, ; , ; Phillips, ; Wilson, M. Can. J . Zool. 1984, 62, 1971-1976. Ivie, G. W.; B u l l , D. L.; Beier, R. C.; Pryor, N. W.; O e r t l i , E. H. Science 1983, 221, 374-376. B u l l , D. L.; Ivie, G. W.; Beier, R. C.; Pryor, N. W.; O e r t l i , E. H. J . Chem. Ecol. 1984, 10, 893-911. Berenbaum, M.; Feeny, P. Science 1981, 212, 927-929. Ivie, G. W.; B u l l , D. L.; Beier, R. C.; Pryor, N. W. J . Chem. Ecol. 1986, 12, 871-884. B u l l , D. L.; Ivie, G. W.; Beier, R. C.; Pryor, N. W. J . Chem. Ecol. 1986, 12, 885-892. Schimmer, O.; Fischer, K. Mutat. Res. 1980, 79, 327-330. Ivie, G. W.; MacGregor, J . T.; Hammock, B. D. Mutat. Res. 1980, 79, 73-77. Monti-Bragadin, C.; Tamaro, M.; Venturini, S.; Pani, B.; Babudri, N.; Baccichetti, F. Farmaco Ed. S c i . 1981, 36, 551-556.

R E C E I V E D January13,1987

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 16

Fungicidal Activity of Naturally Occurring Photosensitizers G. H. Neil Towers and Donald E. Champagne Department of Botany, University of British Columbia, Vancouver, British Columbia V6T 2B1, Canada

In recent years a with in v i t r o f u n g i c i d a Many type I photosensitizer photobin DNA, g linear and angular furanocoumarins, furanochromones, furanoquinolines, and probably the ß-carboline a l k a l o i d s . Isoflavonoids and a l i p h a t i c polyacetylenes attack membrane targets v i a a free r a d i c a l mechanism. Aromatic polyacetylenes display competing type I and type II reactions and the thiophenes are strictly photodynamic s e n s i t i z e r s . The evidence f o r these toxic mechanisms i s discussed, and evidence f o r the involvement of these phytochemicals resistance to fungal attack i n vivo i s reviewed. D a n i e l s (J_) has d e s c r i b e d a s i m p l e and economic p r o c e d u r e which p e r m i t s t h e r a p i d s c r e e n i n g o f p l a n t s and p l a n t e x t r a c t s f o r photot o x i c a c t i v i t y . T h i s t e c h n i q u e was o r i g i n a l l y used t o i d e n t i f y f u r a nocoumarins as t h e compounds r e s p o n s i b l e f o r t h e p h o t o s e n s i t i z i n g action of various umbelliferous plants. More r e c e n t l y , an ever increasing number o f p h o t o t o x i c secondary m e t a b o l i t e s , i n c l u d i n g a l k a l o i d s , p h e n o l i c s , q u i n o n e s , t e r p e n o i d s , and a c e t y l e n e s and t h e i r t h i o p h e n e d e r i v a t i v e s have been i s o l a t e d from v a s c u l a r p l a n t s , f u n g i and b a c t e r i a . As y e a s t s , ( p a r t i c u l a r l y Candida, Saccharomyces, and R h o d o t o r u l a ) , and o t h e r f u n g i , a r e used i n these a s s a y s , most o f t h e known p h o t o s e n s i t i z e r s a r e f u n g i c i d a l , a l t h o u g h i t i s n o t always c l e a r t h a t such a c t i v i t y r e f l e c t s t h e r o l e o f t h e s e compounds i n t h e plant. P h o t o s e n s i t i z e r s v a r y i n b o t h t h e i r mechanisms o f a c t i o n and t a r g e t s i t e s w i t h i n the c e l l . TJie two mechanisms r e c o g n i z e d , termed t y p e I and type I I , a r e reviewed b y C S . Foote elsewhere i n t h i s volume and w i l l be d e s c r i b e d o n l y b r i e f l y h e r e . In b o t h type I and t y p e I I r e a c t i o n s the g r o u n d - s t a t e s e n s i t i z e r i s p h o t o e x c i t e d t o t h e u n s t a b l e s i n g l e t s t a t e , f o l l o w e d by i n t e r s y s t e m c r o s s i n g t o y i e l d the l o n g e r - l i v e d t r i p l e t s e n s i t i z e r (2^_3). i n type I r e a c t i o n s t h e triplet sensitizer participates i n radical or electron transfer r e a c t i o n s w i t h s u s c e p t i b l e b i o m o l e c u l e s , thus consuming t h e s e n s i tizer. Type I I s e n s i t i z a t i o n s i n v o l v e t h e t r a n s f e r o f e x c i t a t i o n

0097-6156/87/0339-0231 $06.00/0 © 1987 American Chemical Society

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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energy from the triplet s e n s i t i z e r to ground-state (triplet) m o l e c u l a r oxygen, r e t u r n i n g the s e n s i t i z e r to i t s ground s t a t e and generating s i n g l e t oxygen. The s e n s i t i z e r may subsequently be r e e x c i t e d and c a t a l y z e f u r t h e r r e a c t i o n s . As t y p e I I s e n s i t i z a t i o n s r e q u i r e the p a r t i c i p a t i o n o f m o l e c u l a r oxygen, the a c t i v i t y o f such p h o t o s e n s i t i z e r s i s a b o l i s h e d under a n a e r o b i c c o n d i t i o n s and can be modified by a z i d e o r D^O, which a l t e r the l i f e t i m e of s i n g l e t oxygen. Some p h o t o s e n s i t i z e r s d i s p l a y an i n t e r m e d i a t e mechanism i n which b o t h t y p e I and type I I r e a c t i o n s o c c u r c o m p e t i t i v e l y . P o t e n t i a l l y p h o t o t o x i c s e c o n d a r y m e t a b o l i t e s a r e known t o o c c u r i n over t h i r t y f a m i l i e s o f v a s c u l a r p l a n t s ; i n some f a m i l i e s a s i n g l e s p e c i e s may elaborate s e v e r a l c l a s s e s of p h o t o s e n s i t i z e r s d e r i v e d from independant b i o s y n t h e t i c r o u t e s (3) . The f u n c t i o n o f t h e s e compounds i s not easy t o e s t a b l i s h , but t h e i r broad-spectrum b i o c i d a l a c t i v i t y a g a i n s t n o t o n l y f u n g i but a l s o b a c t e r i a and i n s e c t s , and t h e i r f r e q u e n t i n v o l v e m e n t i n p h y t o a l e x i n responses, s t r o n g l y suggests t h a t the functio t f generalized d e f e n s e a g a i n s t pathogen p r e c l u d e the p o s s i b i l i t m e t a b o l i s m as w e l l . Type I

Photosensitizers

Type I p h o t o s e n s i t i z e r s may c o v a l e n t l y b i n d t o a v a r i e t y o f s u s c e p t i b l e t a r g e t m o l e c u l e s , i n c l u d i n g p r o t e i n s and tRNA ( / but the m a j o r i t y appear t o form ad d u c t s with DNA, and so may be termed photogenotoxic (9-11) . Such compounds are typically planar, t r i c y c l i c molecules. The b e s t known and f i r s t d e s c r i b e d o f the p h o t o g e n o t o x i n s a r e the furanocoumarins, c h a r a c t e r i s t i c secondary metabolites of the Rutaceae, Apiaceae ( U m b e l l i f e r a e ) , and c e r t a i n other f a m i l i e s of flowering plants. Daniels (_1_) f i r s t showed t h a t f u r a n o c o u m a r i n s cause l e t h a l damage t o y e a s t s i n l i g h t , and t h i s was subsequently c o n f i r m e d i n numerous studies with y e a s t s and other fungi (1220) . T o x i c i t y r e s u l t s m a i n l y from p h o t o b i n d i n g to the pyrimidine b a s e s o f DNA by means o f double bonds a t the 3 , 4 and 4 ' , 5 ' sites, forming monoadducts (21,22) or, in the case of some linear psoralens, b i f u n c t i o n a l adducts leading to interstrand crosslinkages (23-27). This photoactivity i s clearly ecologically relevant, as furanocoumarins are involved in the phytoalexin r e s p o n s e t o f u n g a l i n f e c t i o n i n some u m b e l l i f e r s ( 2 8 - 3 0 ) and these compounds can p h o t o s e n s i t i z e i n s e c t s and o t h e r h e r b i v o r e s (3J_) • Other compounds which d i s p l a y t h i s type o f a c t i v i t y a r e the furanochromones (32) , furanochromenes (33), furanoquinolines, and certain tryptophan-derived a l k a l o i d s i n c l u d i n g the p-carbolines (34-36)• The best u n d e r s t o o d o f t h e s e are the furanoquinoline a l k a l o i d s , p a r t i c u l a r l y d i c t a m n i n e ( I ) , which o c c u r i n a number o f r u t a c e o u s s p e c i e s i n c l u d i n g Skimmia j a p o n i c a and Dictamnus a l b u s (37) . D i c t a m n i n e , skimmianine ( I I ) , m a c u l o s i d e , and m a c u l i n e were phototoxic to the yeasts Saccharomyces c e r e v i s i a e and Candida a l b i c a n s i n UVA (36); d i c t a m n i n e was a l s o p h o t o t o x i c to filamentous p h y t o p a r a s i t i c and z o o p a r a s i t i c f u n g i i n c l u d i n g Mucor h i e m a l i s , M. rammanianus, Fusariurn g r a m i n e a r u s , and P e n i c i l l i u m i t a l i c u m (38) . Both d i c t a m n i n e and skimmianine i n h i b i t e d m i t o s i s and caused g r o s s

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TOWERS A N D C H A M P A G N E

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chromosomal changes t o C h i n e s e hamster o v a r y (CHO) c e l l s i n UVA but n e t i n the d a r k , s u g g e s t i n g a c e l l u l a r t a r g e t i n the n u c l e u s , s i m i l a r t o the f u r a n o c o u m a r i n s (39^) • S u b s e q u e n t l y i t was shown t h a t [ H ] - d i c t a m n i n e i n t e r c a l a t e s w i t h c a l f - t h y m u s DNA i n the d a r k , and a f t e r near-UV i r r a d a t i o n and g e l f i l t r a t i o n t o s e p a r a t e the unbound a l k a l o i d , the l a b e l was found t o be bound t o the DNA (40) . H y d r o x y a p a t i t e chromatography o f h e a t - d e n a t u r e d [ H]-dictamnine-DNA complex showed o n l y s i n g l e - s t r a n d e d DNA, i n d i c a t i n g the f o r m a t i o n o f monoadducts. S t u d i e s o f the p h o t o b i n d i n g o f d i c t a m n i n e towards v a r i o u s s y n t h e t i c DNA's showed t h a t the r a t i o o f b i n d i n g t o p o l y ( d A dT) . p o l y ( d A - d T ) :poly(dG-dC) • p o l y ( dG-dC) :poly( dA-dU) . p o l y (dA-U):p o l y ( d A ) • p o l y ( d T ) , i n r e l a t i o n t o t h a t o f c a l f thymus DNA, was 18:1:0.5:0.3/ s i m i l a r t o the r a t i o o b s e r v e d f o r the f u r a n o c o u m a r i n 8-methoxypsoralen (8-MOP). In a d d i t i o n , p r i o r t r e a t m e n t o f DNA w i t h d i c t a m n i n e g r e a t l y r e d u c e d i n c o r p o r a t i o n o f 8-MOP, s u g g e s t i n g t h a t the b i n d i n g s i t e s f o r the two compounds a r e p r o b a b l y i d e n t i c a l . Template a c t i v i t y o f th measured by the RNA polymeras c a l f thymus DNA was l e s y 3

3

3

When c u l t u r e s o f Mucor h i e m a l i s were i n c u b a t e d with [ H]d i c t a m n i n e i n the l i g h t , 0.2% o f the a d m i n i s t e r e d l a b e l (0.18 ug-mg DNA" ) was i n c o r p o r a t e d i n t o the f u n g a l DNA i n v i v o (38) • The c h e m i s t r y o f the c o v a l e n t adducts o f dictamnine with n u c l e i c a c i d b a s e s has n o t been d e s c r i b e d . These f u r a n o q u i n o l i n e s may have a r o l e i n p r o t e c t i n g some p l a n t s a g a i n s t f u n g a l a t t a c k , but t h i s has y e t t o be demonstrated. The furanochromones khellin ( I I I ) and v i s n a g i n , t h e active p r i n c i p a l s o f the m e d i c i n a l p l a n t Ammi v i s n a g a ( 4 1 ) , a r e p h o t o t o x i c towards g r a m - p o s i t i v e b a c t e r i a (32/42)/ v i r u s e s (43) / and Saccharomyces and Candida ( 3 2 ) . K h e l l i n i n d u c e s m e l a n i z a t i o n i n r a b b i t s k i n i n s u n l i g h t (4£) , and causes g r o s s chromosomal damage i n CHO c e l l s (32) . Longwave UV i r r a d i a t i o n o f a f r o z e n aqueous s u s p e n s i o n o f k h e l l i n and thymine r e s u l t e d i n the f o r m a t i o n o f a 2-2 adduct (IV) between the 2,3 double bond o f k h e l l i n and the 5*,6* double bond o f thymine, i n d i c a t i n g t h a t the p h o t o t o x i c i t y o f k h e l l i n i s due t o a mechanism s i m i l a r t o the f u r a n o c o u m a r i n s . The incorporation of furanochromones i n t o DNA i n v i v o has y e t t o be d e m o n s t r a t e d . Some furanochromones, i n c l u d i n g k h e l l i n / a r e a l s o known t o be insect antifeedants (45) , but as y e t we can o n l y s p e c u l a t e about the ecological significance of their phototoxic a c t i v i t y . S i m i l a r l y / t h e r o l e o f p h o t o t o x i c i t y i n the a c t i v i t y o f the p o t e n t c a r c i n o g e n s , the a f l a t o x i n s ( V a , b ) , p r o d u c e d by A s p e r g i l l u s f l a v u s and r e l a t e d s p e c i e s (46) i s n o t a p p a r e n t . A f l a t o x i n s are p h o t o t o x i c t o Paramecium but n o t t o E* c o l i (47) , and have n o t y e t been t e s t e d f o r p h o t o t o x i c i t y a g a i n s t f u n g i . In the mammalian l i v e r (and hence i n the dark) t h e y a r e c o n v e r t e d t o h e p a t o c a r c i n o g e n s when t h e double bond o f the f u r a n r i n g i s e p o x i d i z e d and the p r o d u c t s u b s e q u e n t l y forms c o v a l e n t a d d u c t s t o DNA (48) . E x c i t a t i o n by UVA (365 nm) a l s o i n d u c e s the f o r m a t i o n o f adducts i n v i t r o ( 4 9 ) . The p - c a r b o l i n e o r harmane a l k a l o i d s ( V I ) , the a l k a l o i d s 6-cant h i n o n e and 5-methoxy-6-canthinone ( a l l from v a r i o u s R i t a c e a e ) and the N-methyl s u b s t i t u t e d harmane b r e v i c o l l i n e from the sedge Car ex b r e v i c o l l i s are p h o t o t o x i c to Saccharomyces and Candida ( 3 4 ) . S t r u c t u r a l s i m i l a r i t y t o the f u r a n o c o u m a r i n s s u g g e s t s t h a t t h e y t o o may 1

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LIGHT-ACTIVATED PESTICIDES

p h o t o b i n d t o DNA; t h i s work i s p r e s e n t l y underway i n our l a b o r a t o r y . The i s o f l a v o n o i d p h y t o a l e x i n s p h a s e o l l i n , 3,6a,9- t r i h y d r o x y p t e r o c a r p a n , g l y c e o l l i n , t u b e r o s i n , and p i s a t i n , p r o d u c e d by s e v e r a l s p e c i e s o f Eabaceae, p h o t o i n a c t i v a t e d g l u c o s e - 6 - p h o s p h a t e dehydro genase i n an in v i t r o a s s a y (50_) • S i n g l e t oxygen q u e n c h e r s , h y d r o x y l r a d i c a l s c a v e n g e r s , and s u p e r o x i d e dismutase d i d n o t p r o t e c t t h e enzyme a g a i n s t p h o t o i n a c t i v a t i o n , r u l i n g out a type I I mechanism. ESR measurements c o n f i r m e d t h e p r o d u c t i o n o f f r e e r a d i c a l s , which were most s t a b l e i n t h e case o f p h a s e o l l i n . In t h e d a r k , g l y c e o l l i n i n h i b i t s e l e c t r o n t r a n s p o r t a t some p o i n t beyond t h e s u c c i n a t e dehydrogenase s i t e ( 5 1 ) , and p i s a t i n appears t o u n c o u p l e o x i d a t i v e phosphorylation (52). The a l i p h a t i c polyacetylenes increase membrane p e r m e a b i l i t y and a r e h i g h l y t o x i c t o Saccharomyces and o t h e r y e a s t s ; t h e i r t o x i c i t y i s not oxygen dependent and t h e i r r a p i d p o l y m e r i z a t i o n i n UVA has been taken as e v i d e n c e o f f r e e r a d i c a l f o r m a t i o n (53) • Some chromenes and b e n z o f u r a n s , i n c l u d i n g e n c e c a l i n (VII), are phototoxic Saccharomyce d Candid (33) Thes compounds cause h e m o l y s i i n the membrane, and ma Intermediate

Photosensitizers

Many p h o t o s e n s i t i z e r s a r e c a p a b l e o f competing type I and type I I reactions. Even 8-MOP, l o n g c o n s i d e r e d a c l a s s i c a l type I p h o t o s e n s i t i z e r , g e n e r a t e s s i n g l e t oxygen i n t h e absence o f s u i t a b l e s i t e s for photobinding (S5). The r e l a t i v e s i g n i f i c a n c e o f t h e two mechanisms i s i l l u s t r a t e d by t h e o b s e r v a t i o n t h a t JE. c o l i mutants d e f e c t i v e i n t h e r e p a i r o f o x i d a t i v e damage a r e about 15 t i m e s l e s s s e n s i t i v e t o 8-MOP i n d u c e d damage than a r e e x c i s i o n r e p a i r d e f i c i e n t m u t a n t s , b u t a r e about 100 t i m e s more s e n s i t i v e than w i l d type s t r a i n s (56). Competing type I and type I I mechanisms a r e t y p i c a l o f t h e aromatic p o l y a c e t y l e n e s , c h a r a c t e r i s t i c secondary m e t a b o l i t e s o f the Asteraceae and about twenty o t h e r families o f vascular plants. E a r l y work on t h e p o l y a c e t y l e n e phenylheptatriyne (PHT) ( V I I I ) showed reduced t o x i c i t y t o Saccharomyces c e r e v i s i a e under a e r o b i c c o n d i t i o n s , and t o x i c i t y was n o t m o d i f i e d by a z i d e (which quenches s i n g l e t oxygen) o r E^O (which i n c r e a s e s t h e l i f e t i m e o f s i n g l e t oxygen) ( 5 7 ) . Low c o n c e n t r a t i o n s o f PHT r a p i d l y i n h i b i t e d cell r e s p i r a t i o n , and d i d n o t i n c r e a s e s i s t e r c h r o m a t i d exchanges ( 5 8 ) , i n d i c a t i n g t h a t t h e n u c l e u s was n o t a t a r g e t ; t h i s compound thus d i d not resemble e i t h e r t h e f u r a n o c o u m a r i n s o r t h e photodynamic dyes i n its mechanism of action. To c o m p l i c a t e the s t o r y , i t was subsequently found t h a t PHT and o t h e r p o l y a c e t y l e n e s a r e p h o t o dynamic towards some o r g a n i s m s , i n c l u d i n g E. c o l i , b u t a r e p a r t i a l l y non-photodynamic i n o t h e r systems, i n c l u d i n g Saccharomyces (53) • With l i p o s o m e s as a model membrane system, o n l y t h e photodynamic a c t i v i t y c o u l d be demonstrated ( 5 9 ) . The e f f e c t o f PHT on membrane p e r m e a b i l i t y was shown t o depend on t h e degree o f u n s a t u r a t i o n o f the membrane lipids. Permeability was g r e a t l y i n c r e a s e d i n l i p o s o m e s composed o f d i p a l m i t o y l p h o s p h a t i d y l c h o l i n e (PC) and o t h e r s a t u r a t e d l i p i d s , which p r e s e n t a h i g h l y o r d e r e d environment, and was i n c r e a s e d t o a l e s s e r e x t e n t i n d i s o r d e r e d membranes composed o f u n s a t u r a t e d l i p i d s ; i n t h e l a t t e r case l i p i d p e r o x i d a t i o n was shown

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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to be i n v o l v e d . The d i f f e r e n t e f f e c t s o f PHT i n d i f f e r e n t o r g a n i s m s may be r e l a t e d t o d i f f e r i n g l i p i d environments i n the membranes, but PHT also inactivates membrane-bound enzymes (60) and so the r e s p o n s e s may a l s o be r e l a t e d t o a c c e s s i b i l i t y o f t a r g e t p r o t e i n s , possibly including respiratory centers. Photochemical s t u d i e s p r o v i d e f u r t h e r i n f o r m a t i o n on competing t y p e I and type I I r e a c t i o n s w i t h PHT. L a s e r e x c i t a t i o n (308 or 337 nm) l e a d s t o the f o r m a t i o n o f a s t r o n g t r i p l e t s i g n a l , w i t h a l i f e t i m e o f 28 us i n m e t h a n o l , which was e f f i c i e n t l y quenched by t h e t r i p l e t quencher 1,3 o c t a d i e n e (61) . Quenching w i t h occurred w i t h a r a t e c o n s t a n t comparable t o the r a t e o f e l e c t r o n t r a n s f e r t o methyl v i o l o g e n . The formation of both s i n g l e t oxygen and the semioxidized PHT radical are c o n s i s t e n t with the competing mechanisms o b s e r v e d in vivo. When i r r a d i a t e d w i t h UVA, liposome b i l a y e r s composed o f d i s t e a r y l PC w i t h PHT p r o d u c e d a f r e e r a d i c a l s i g n a l d e t e c t e d by e l e c t r o n s p i n r e s o n a n c e (ESR) s p e c t r o s c o p y ( 6 2 ) • The spectrum c o n s i s t e d and a g v a l u e o f 2.0017 light intensity, PHT bilayer, concentration of the liposome-PHT s u s p e n s i o n . The signal was enhanced i n an a n a e r o b i c environment, i n d i c a t i n g a n o n - o x i d a t i v e mechanism f o r f r e e r a d i c a l f o r m a t i o n . Cnce formed, the radical s p e c i e s was v e r y s t a b l e i n the p r e s e n c e o f oxygen, d e c a y i n g s l o w l y o v e r an 8-12 hour p e r i o d . F o r m a t i o n o f the r a d i c a l was enhanced i n an o r d e r e d l i p i d environment as i n c o r p o r a t i o n o f l y s o p h o s p h a t i d y l c h o l i n e , which p e r t u r b s l i p i d p a c k i n g , d e c r e a s e d l e v e l s o f the f r e e radical species. When PHT was p r e s e n t i n a l i p o s o m e w i t h an even more f l u i d membrane, such as egg y o l k PC, the l e v e l s o f f r e e r a d i c a l g e n e r a t i o n were even l o w e r . P o l y a c e t y l e n e involvement i n r e s i s t a n c e to fungal a t t a c k i s well d o c u m e n t e d , and i n c l u d e s the phytoalexins safynol and d e h y d r o s a f y n o l from Carthamnus t i n i c t o r i u s (63^,64), wyerone from the broad bean, V i c i a faba (65) , and falcarinol, falcarindiol, and E - t e t r a d e c a - 6 - e n e - 1 , 3 - d i y n e - 5 , 8 - d i o l from the tomato, L y c o p e r s i c o n e s c u l e n t u m (66) • These p h y t o a l e x i n s a r e t o x i c w i t h o u t p h o t o a c t i v a t i o n , but t h e i r a c t i v i t y may, i n some c a s e s , be enhanced i n UVA. Few s t u d i e s s p e c i f i c a l l y a d d r e s s the r o l e o f p o l y a c e t y l e n e p h o t o s e n s i t i z a t i o n i n defense a g a i n s t f u n g a l a t t a c k . PHT, p r e s e n t i n the c u t i c l e o f Bidens p i l o s a l e a v e s a t c o n c e n t r a t i o n s up t o 600 ppm, s t r o n g l y i n h i b i t s the g e r m i n a t i o n and growth o f F u s a r i u m culmorum i n UVA but not i n the d a r k ; PHT was f u n g i t o x i c and not s i m p l y f u n g i s t a t i c (67). In t h i s case the p o l y a c e t y l e n e c o n s t i t u t e s a p r e f o r m e d barrier against fungal attack. Nineteen species of phylloplane yeasts and yeast-like fungi, isolated from Hawaiian s p e c i e s o f B i d e n s w i t h and w i t h o u t l e a f p o l y a c e t y l e n e s , were t e s t e d f o r p h o t o s e n s i t i v i t y t o those a c e t y l e n e s (68) • A l t h o u g h a l l t h e s e o r g a n i s m s , members of the Sfcorobolmycetaceae, Cryptococcaceae, and E\ingi Imperfect!, were s e n s i t i v e t o some a c e t y l e n e s and r e s i s t a n t t o o t h e r s , t h e r e was no c o r r e l a t i o n between the p r e s e n c e o r absence o f leaf polyacetylenes and the distribution of these saprophytes amongst the B i d e n s s p e c i e s , w i t h one n o t a b l e e x c e p t i o n . The o n l y p a t h o g e n i c s p e c i e s i s o l a t e d , C o l l e t o t r i c h u m g l o e o s p o r i o d e s , d i d not c o l o n i z e B i d e n s l e a v e s c o n t a i n i n g C13 a r o m a t i c p o l y a c e t y l e n e s , t o which i t i s e x t r e m l y s e n s i t i v e i n v i t r o .

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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236 Type I I P h o t o s e n s i t i z e r s

The c l a s s i c type I I s e n s i t i z e r s a r e the photodynamic dyes; t h e most active natural type II s e n s i t i z e r s yet d i s c o v e r e d are the thiophenes, sulfur derivatives of the polyacetylenes. These compounds a r e r e s t r i c t e d t o advanced t r i b e s o f the A s t e r a c e a e , including the Vernonieae, Inuleae, Heliantheae, Anthemideae, S e n e c i o n e a e , Cynareae, and p a r t i c u l a r l y t h e Tageteae (69) . Alphat e r t h i e n y l (a-T) (IX) i s t o x i c t o b a c t e r i a , y e a s t s , and o t h e r f u n g i ( 1 1 ) , and can p h o t o s e n s i t i z e h e r b i v o r o u s i n s e c t s ( 7 0 , 7 1 ) . A strict r e q u i r e m e n t f o r oxygen i n a-T t o x i c i t y has been demonstrated b o t h i n vivo (60,72-74) and in vitro (75-77) . Although the specific c e l l u l a r t a r g e t i n v o l v e d has n o t been i d e n t i f i e d , i t i s c e r t a i n l y i n t h e membrane as a-T i s not p h o t o g e n o t o x i c , and i t i n c r e a s e s membrane p e r m e a b i l i t y and i n a c t i v a t e s membrane-bound enzymes (60,74)• The membrane-bounded v i r u s e s murine c y t o m e g a l o v i r u s (CMV) and SLndbis v i r u s were s e n s i t i v e t T t 10 l i UVA but t i th dark b u t the membraneless v i r u CMV which had been i n a c t i v a t e e f f i c i e n t l y but the v i r a l DNA c o u l d not r e p l i c a t e and l a t e r v i r a l p r o t e i n s were n o t p r o d u c e d . As v i r a l gene e x p r e s s i o n was i n h i b i t e d i t was s u g g e s t e d t h a t a-T may i n t e r a c t w i t h v i r a l p r o t e i n s as w e l l as membrane l i p i d s ; a-T c e r t a i n l y o x i d i z e s p r o t e i n s i n IS. c o l i (74) • The t h i o p h e n e s a r e i n v o l v e d i n d e f e n s e a g a i n s t f u n g a l a t t a c k i n Tagetes a t l e a s t . I n n o c u l a t i o n o f T a g e t e s e r e c t a w i t h the pathogen Fusarium oxysporum e l i c i t s t w e l v e - f o l d g r e a t e r p r o d u c t i o n o f a-T, b i t h i o p h e n e h y d r o x y l , and b i t h i o p h e n e a c e t a t e (19) . Two s t r a i n s o f t h e pathogen a r e known; t h e f a s t - g r o w i n g v i r u l e n t s t r a i n k i l l s t h e p l a n t b e f o r e s i g n i f i c a n t e l e v a t i o n o f the t h i o p h e n e l e v e l s can be a c c o m p l i s h e d , but t h e s l o w e r - g r o w i n g s t r a i n i s e f f e c t i v e l y combatted by t h e s e f u n g i c i d a l compounds. R e c e n t l y we have d e s c r i b e d the a n t i f u n g a l a c t i v i t y o f a group o f a c e t y l e n i c d i t h i a c y c l o h e x a d i e n e s , from C h a e n a c t i s d o u g l a s i i and o t h e r members o f t h e Asteraceae (80). These r e d compounds, c h r i s t e n e d t h i a r u b r i n e s ( X ) , do n o t r e q u i r e l i g h t f o r t h e i r a n t i fungal a c t i v i t y . However, when i r r a d i a t e d w i t h UVA, t h e i r a c t i v i t y i s enhanced and i s t h e n extended t o b a c t e r i a and v i r u s e s . In l i g h t t h e s e u n s t a b l e compounds l o s e one o f the s u l f u r atoms o f t h e r i n g and the r e s u l t a n t t h i o p h e n e then d i s p l a y s the photodynamic a c t i v i t y c h a r a c t e r i s t i c of t h i s c l a s s of phytochemicals. The dark a n t i f u n g a l a c t i v i t y remains u n e x p l a i n e d . The activity o f the fungal photosensitizer cercosporin, a d i h y d r o x y p e r y l e n e quinone produced by v a r i o u s C e r c o s p o r a s p e c i e s , i s r e v i e w e d by Daub e l s e w h e r e i n t h i s volume. This apparently c o n s t i t u t e s t h e f i r s t case i n which a f u n c t i o n o t h e r than d e f e n s e can be a s c r i b e d t o a p h o t o t o x i n . As C e r c o s p o r a hyphae do n o t p e n e t r a t e the c e l l s o f the h o s t p l a n t , c e r c o s p o r i n - m e d i a t e d l i p o p e r o x i d a t i o n o f h o s t membranes i s e s s e n t i a l t o r e l e a s e n u t r i e n t s r e q u i r e d by the growing pathogen. Hypericin, a related compound found i n most s p e c i e s o f Hypericum, i s a l s o a photodynamic p h o t o s e n s i t i z e r (81, P. R i o x , t h i s v o l u m e ) . The p h o t o t o x i n o c c u r s i n g l a n d u l a r t r i c h o m e s and i s e f f e c t i v e a g a i n s t i n s e c t s , but i t s s i g n i f i c a n c e as a p o s s i b l e a n t i f u n g a l compound i s unknown.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

TOWERS AND CHAMPAGNE

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S t r u c t u r e s I-X

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Conclusion The d i v e r s i t y o f p h o t o t o x i c compounds i s o l a t e d from p l a n t s , f u n g i , and b a c t e r i a i n r e c e n t y e a r s s u g g e s t s t h a t such compounds may be q u i t e common i n n a t u r e . P r o b a b l y hundreds o f such compounds remain t o be i d e n t i f i e d . Much work remains t o be done t o i d e n t i f y t h e t o x i c mechanism and t a r g e t s i t e s o f many o f t h e p h o t o s e n s i t i z e r s which a r e c u r r e n t l y known. The q u e s t i o n o f how o r g a n i s m s t h a t p r o d u c e p h o t o t o x i n s a v o i d a u t o t o x i c i t y has s c a r c e l y been a d d r e s s e d . Finally, the ecological significance o f phototoxic secondary m e t a b o l i t e s has o n l y begun t o be s t u d i e d , b u t g i v e n t h e d i v e r s i t y o f s p e c i e s which c o n t a i n t h e s e compounds many f a s c i n a t i n g i n t e r a c t i o n s await d e s c r i p t i o n .

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239

22. Musajo, J.F.; Bordin, F,; Caporale, G.; Marciani, S.; Rigatti, G. Photochem. Photobiol. 1967, 6, 711-719. 23. Cole, R.S. Biochem. Biophys. Acta 1970, 217, 30-39. 24. Cole, R.S. J . Bacteriol. 1971, 107, 846-852. 25. Chandra, P.; Kraft, S.; Wacker, A . ; Rodighiero, G.; Dall'Acqua, F . ; Marciani, S. Biophysik. 1971, 7, 251- 258. 26. Marciani, S.; Terbojevich, M.; Dall'Aqcqua, F. Z. Naturforsch. 1972, 27b, 196-200. 27. Musajo, L.; Rodighiero, G. In "Photophysiology, Vol. VII"; Geise, A.C., Ed.; Academic: New York, 1972, pp. 115-147. 28. Johnson, C.; Brannon, D.R. Phytochem. 1973, 12, 2961. 29. Beier, R.C.; Oertli, E.H. Phytochem. 1983, 22, 2595. 30. Chandhary, S.K.; Ceska, O.; Warrington, P.J.; Ashwood-Smith, M.J. J . Agric. Food Chem. 1985, 33, 1153. 31. Berenbaum, M.R. Science 1978, 201, 532-534. 32. Abeysekera, B.F.; Abramowski, Z.; Towers, G.H.N. Photochem. Photobiol. 1983, 38 33. Proksch, P.; Proksch Prod. 1983, 46, 331-335 34. Towers, G.H.N.; Abramowski, Z. J . Nat. Prod. 1983, 46, 576-581. 35. McKenna, D.J.; Towers, G.H.N. Phytochem. 1981, 20. 1001-1004. 36. Towers, G.H.N.; Graham, E.A.; Spenser, I.D.; Abramowski, Z. Planta Medica 1981, 41, 136-142. 37. Murray, R.D.H.; Mendez, J.; Brown, S.A. "The Natural Coumarins: Occurrence, Chemistry, and Biochemistry"; Wiley: New York, 1982. 38. Pfyffer, G.E.; Towers, G.H.N. Can. J . Microbiol. 1982, 28, 468-473. 39. Philogene, B.J.R.; Arnason, J.T.; Towers, G.H.N.; Abramowski, Z.; Campos, F.; Champagne, D.; McLachlan, D. J. Chem. Ecol. 1984, 10, 115-123. 40. Pfyffer, G.A.; Pfyffer, B.U.; Towers, G.H.N. Photochem. Photobiol, 1982, 35, 793-797. 41. Schonberg, A.; Sina, A.J. J. Am. Chem. Soc. 1950, 72, 1611. 42. Fowlks, W.L.; Griffith, D.G.; Oginsky, E.L. Nature 1958, 181, 571-572. 43. Cassuto, E.; Gross, N.; Bardwell, E.; Howard-Flanders, P. Biochem. Biophys. Acta 1977, 475, 589-600. 44. Kabilov, N.M. Farmakol. i. Tokisol. 1962, 25, 733-735. 45. Yajima, T.; Munakata, K. Agric. Biol. Chem. 1979, 43, 1701-1706. 46. Diener, U.L.; Davis, N.D.; In "Aflatoxins"; Goldblatt, L . A . , Ed.; Academic Press: New York, 1969, pp. 13-54. 47. Smith, R.C.; Neely, W.C. Can. J. Microbiol. 1972, 18, 1965-1967. 48. Wogan, G.N. Ann. Rev. Pharmacol. 1975, 15, 437-453. 49. Sheih, J.-C.; Song, P.-S. Cancer Res. 1980, 40, 68950. Bakker, J.; Gommers, F.J.; Smits, L.; Fuchs, A.; de Vries, F.W. Photochem. Photobiol. 1983, 38, 323-330. 51. Kaplan, D.T.; Keen, N.T.; Thomason, I . J . Physiol. Plant Pathol. 1980, 16, 319-325. 52. Oku, H.; Ouchi, S.; Shiraishi, T.; Utsumi, K.; Seno, S. Proc. Jpn. Acad. 1976, 52, 33-36. 53. McLachlan, D.; Arnason, J.T.; Lam, J . Photochem. Photobiol. 1984, 39, 177-182. 54. Aregullin-Gallardo, M. Ph.D. Thesis, University of California, Irvine, 1985.

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55. Vedaldi, D.; Dall Acqua, F.; Gennaro, A . ; Rodighiero, R. Z. Naturforsch. 1983, 38, 866-869. 56. Tuveson, R.W.; Berenbaum, M.R.; Heininger, E.E. J. Chem. Ecol. 1986, 12, 933-948. 57. Arnason, J.T.; Wat, C.-K.; Downum, K.; Yamamoto, E.; Graham, E.; Towers, G.H.N. Can. J. Microbiol. 1980 26, 698-705. 58. MacRae, W.D.; Chan, G.F.Q.; Wat, C.-K.; Towers, G.H.N.; Lam, J . Experientia 1980, 36, 1096-1097. 59. McRae, D.G.; Yamamoto, E.; Towers, G.H.N. Biochem. Biophys. Acta 1985, 821, 488-496. 60. Yamamoto, E.; Wat, C.-K.; MacRae, W.D.; Towers, G.H.N. FEBS Letters 1979, 107, 134-136. 61. Weir, D.; Scaiano, J.C.; Arnason, J.T.; Evans, C. Photochem. Photobiol. 1984, 42, 223-230. 62. McRae, D.; Yamamoto, E.; Towers, G.H.N. (unpublished results). 63. Allen, E.; Thomas, C. Phytochem. 1971, 10, 1579-1582. 64. Allen, E.; Thomas C Phytopathol 1971 61 1107 1109 65. Hargraves, J.A.; Mansfield Phytochem. 1976, 15, 66. DeWit, P.J.G.M.; Koddle, E. Physiol. Plant Pathol. 1981, 18, 143-148. 67. Bourque, G.; Arnason, J.T.; Madhosingh, C.; Orr, W. Can. J. Bot. 1985, 63, 899-902. 68. Marchant, Y.Y.; Towers, G.H.N. Biochem. System. Ecol. (in press). 69. Bohlman, F.; Burkhardt, T.; Zdero, C. "Naturally Occurring Acetylenes"; Academic Press: New York, 1973, 547 p. 70. Downum, K.R.; Rosenthal, G.A.; Towers, G.H.N. Pest. Biochem. Physiol. 1984, 22, 104-109. 71. Champagne, D.E.; Arnason, J.T.; Philogene, B.J.R.; Morand, P. Lam, J . J . Chem. Ecol. 1986, 12, 835-858. 72. Arnason, J.T.; Chan, G.F.Q.; Wat, C.-K.; Downum, K.; Towers, G.H.N. Photochem. Photobiol. 1981, 33, 821- 824. 73. Gommers, F.J.; Bakker, J.; Wynberg, H. Photochem. Photobiol. 1982, 35, 615-619. 74. Downum, K.R.; Hancock, R.E.W.; Towers, G.H.N. Photochem. Photobiol. 1982, 36, 517-524. 75. Wat, C.-K.; MacRae, W.D.; Yamamoto, E.; Towers, G.H.N.; Lam, J . Photochem. Photobiol. 1980, 32, 167-172. 76. Reyftmann, J.P.; Kagan, J.; Santus, R.; Marliere, P. Photochem. Photobiol. 1985, 41, 1-7. 77. Evans, C.; Weir, D.; Scaiano, J.C.; MacEachern, A.; Arnason, J.T.; Morand, P.; Hollebone, B.; Leitch, L.C.; Philogene, B.J.R. Photochem. Photobiol. (in press). 78. Hudson, J.B.; Graham, E.A.; Micki, N.; Hudson, L.; Towers, G.H.N. Photochem. Photobiol. (in press). 79. Kourany, E. M.Sc. Thesis, University of Ottawa, 1986. 80. 80. Towers, G.H.N.; Abramowski, Z.; Finlayson, A.J.; Zucconi, A. Planta Medica 1985, xx, 225-229. 81. Knox, J.P.; Dodge, A.D. Plant, Cell, Environ. 1985, 8, 19-25. RECEIVED December2,1986

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 17

Structure and Function Relationships in Polyacetylene Photoactivity Y. Yoke Marchant and Geoffrey K. Cooper ARCO Plant Cell Research Institute, 6560 Trinity Court, Dublin, CA 94568

Polyacetylenes and thiophene hav light-independent d photoactivated toxi These b i o l o g i c a l l y activ practical interest because of their possible role as plant defense compounds and their potential as commercially useful biocidal agents. Many factors influence the extent of damage caused by these compounds and this paper reviews the relationship between structure and function in polyacetylenes and presents data in support of current views.

P o l y a c e t y l e n e s and t h e i r thiophene d e r i v a t i v e s are b i o l o g i c a l l y a c t i v e secondary m e t a b o l i t e s c h a r a c t e r i s t i c o f t a x o n o m i c a l l y advanced p l a n t f a m i l i e s such as the A s t e r a c e a e , the A p i a c e a e , the A r a l i a c e a e and the Campanulaceae, a s w e l l as c e r t a i n groups o f Basidiomycete f u n g i ( 1 - 5 ) . Only seven compounds were d e s c r i b e d between 1902, when Arnaud f i r s t e s t a b l i s h e d the e x i s t e n c e o f a n a t u r a l l y - o c c u r r i n g t r i p l e bond (6), and 1950, when a n t i b i o t i c substances produced by f u n g a l s p e c i e s were i d e n t i f i e d a s a c e t y l e n e s (7-12; F i g u r e 1). S i n c e then s e v e r a l hundred p o l y a c e t y l e n e s have been recorded ( 3 ) , many w i t h t o x i c a c t i v i t y a g a i n s t b i o l o g i c a l systems, because i n v e s t i g a t i o n s i n t o the a n t i b i o t i c p r o p e r t i e s o f p l a n t s and f u n g i o f t e n l e d t o the d i s c o v e r y o f p o l y a c e t y l e n e s a s the active principles. I d e n t i f i c a t i o n was f a c i l i t a t e d by the c h a r a c t e r i s t i c UV s p e c t r a o f conjugated a c e t y l e n e s and by the h i g h e x t i n c t i o n c o e f f i c i e n t s o f the s p e c t r a which p e r m i t t e d d e t e c t i o n o f low q u a n t i t i e s o f compounds i n e x t r a c t s (1,13,14). In 1973 the n e m a t o c i d a l p r o p e r t i e s o f a l p h a - t e r t h i e n y l ( I I ) and 5 - ( 3 - b u t e n - 1 - y n y l ) - 2 , 2 ' - b i t h i e n y l ( I I I ; F i g u r e 2) were found t o be s i g n i f i c a n t l y enhanced by UV l i g h t ( 1 5 ) . T h i s l e d to a s y s t e m a t i c i n v e s t i g a t i o n o f the p h o t o t o x i c p r o p e r t i e s o f p o l y a c e t y l e n e s from the Asteraceae by Towers and h i s a s s o c i a t e s ( 5 , 16-19). The in. v i t r o p h o t o a c t i v i t y o f a c e t y l e n i c compounds a g a i n s t b i o l o g i c a l systems i s now a w e l l e s t a b l i s h e d phenomenon. N e v e r t h e l e s s , many b i o l o g i c a l l y a c t i v e a c e t y l e n e s are not l i g h t - a c t i v a t e d and must be c o n s i d e r e d i n any attempt to understand the s t r u c t u r a l b a s i s f o r the p h o t o t o x i c i t y o f these compounds. T h i s paper w i l l examine the 0097-6156/87/0339-0241 $06.00/0 © 1987 American Chemical Society

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r e l a t i o n s h i p between s t r u c t u r e and b i o l o g i c a l a c t i v i t y i n polyacetylenes. Polyacetylenes with Light-Independent B i o l o g i c a l E f f e c t s Bohlmann e t a l . (1) surveyed 32 s p e c i e s i n the Campanulaceae and found t h a t the most commonly o c c u r r i n g a c e t y l e n e s i n t h i s f a m i l y a r e the CiH-ene-diyne-ene t e t r a h y d r o p y r a n y l e t h e r s ( I V ; F i g u r e 3 ) , none o f which appear to be p h o t o s e n s i t i z e r s , or even to have a n t i b i o t i c a c t i v i t y ( 5 ) . The c h a r a c t e r i s t i c compounds o f the Apiaceae and the A r a l i a c e a e , f a l c a r i n o n e and f a l c a r i n d i o l (V, V I ; Table I ) , a r e T a b l e I. B i o l o g i c a l l y a c t i v e C17 a c e t y l e n e s from the Apiaceae and A r a l i a c e a e

CH =CH-C-(C=C) -CH -CH=CH-(CH ) -CH 2

2

2

2 6

3

OH CH=CH - C - (C=C) -CH -CH-CH -(CH ) -CH OH 2

2

2

2 6

3

OH CH -(CH ) -CH-(CH ) -(CH=CH) -(C=C) -CH=CH-(CH ) 0H 3

2 2

2 2

2

2

2

2

CH-(CH)-CH-(CH=CH)-(C=C)-CHCHCHOH OH YID 3

2 2

3

2

2

2

2

F a l c a r i n o n e ( V ) , f a l c a r i n d i o l ( V I ) , Daucus c a r o t a L.; o e n a n t h o t o x i n ( V I I ) , Oenanthe c r o c a t a L.; c i c u t o t o x i n ( V I I I ) , C i c u t a v i r o s a L.

a c t i v e a g a i n s t pathogenic and d e r m a t o p h y t i c f u n g i (20-27), i n s e c t s such as Daphnia magna S t r a u b ( 2 8 ) , cause e r y t h r o c y t e h e m o l y s i s (21,29), and have v a r i o u s p h a r m a c o l o g i c a l e f f e c t s ( 3 0 ) , a l l o f which are l i g h t independent. E f f e c t i v e c o n c e n t r a t i o n s d i f f e r f o r each e x p e r i m e n t a l system, but appear to be w i t h i n the range o f 10-5 t o 10-4 M. Two o t h e r C17 p o l y a c e t y l e n e s , o e n a n t h o t o x i n ( V I I ) and c i c u t o t o x i n ( V I I I ) from Oenanthe c r o c a t a L. and C i c u t a v i r o s a L., are much more potent compounds, w i t h w e l l documented f a t a l n e u r o t o x i c e f f e c t s on l i v e s t o c k and humans ( 3 1 ) . A c e t y l e n e s from the Asteraceae a l s o have numerous nonp h o t o a c t i v a t e d b i o l o g i c a l e f f e c t s (Table I I ) . For example, the

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

17.

M A R C H A N T A N DCOOPER

Polyacetylene Photoactivity

H(C=C) -CH=C=CH-(CH=CH) -CH COOH 2

2

2

F i g u r e 1. The f i r s t f u n g a l a n t i b i o t i c a c e t y l e n e i s o l a t e d from B a s i d i o m y c e t e s . Mycomycin ( I ) , N o c a r d i a a c i d o p h i l u s .

^MgVc^C-CH^H,

F i g u r e 2. P h o t o a c t i v a t e d n e m a t i c i d a l t h i o p h e n e s from t h e Asteraceae. A l p h a - t e r t h i e n y l ( I I ) ; 5-(3-buten-1-ynyl)-2,2' b i t h i e n y l ( I I I ) , T a g e t e s p a t u l a L.

H0CH — CH=CH — (C = C v*/ ) -CH = C H - 0 2 2

2

17

F i g u r e 3. T y p i c a l p o l y a c e t y l e n e from t h e Campanulaceae. Cm-tetrahydropyranylether ( I V ) , Campanula s p p .

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243

244

LIGHT-ACTIVATED PESTICIDES Table I I . Non-photoactivated polyacetylenes from the Asteraceae

CH -(C=C) , /-CH 3 - v=^ C H - k ^ 3

3

R«H K R * CH3CO X 0
3

XI

HC=C-C-C=CH

6 xn CH - CH = CH - (C=C) - CH=CH - COCH 3

2

3

xm

Ichthyothereol (IX), ichtyothereol acetate (X), Ichthyothere terminalis Spreng.; c a p i l l i n (XI), Artemisa c a p i l l a r i s Thunb.; 3methyl-3-phenyl-1,4-pentadiyne (XII), Artemesia monosperma D e l i l e ; matricaria ester (XIII), Solidago altissima L.

a c t i v e p r i n c i p l e s i n the p l a n t s used a s f i s h poisons i n p a r t s o f South America ( I c h t h y o t h e r e and C l i b a d i u m spp.) a r e the Cl4-enet r i y n e - t e t r a h y d r o p y r a n e s i c h t h y o t h e r e o l and i t s a c e t a t e ( I X , X; 3 2 ) , which d i f f e r from t h e i n a c t i v e compounds o f Campanula by t h e presence o f one e x t r a a l k y n y l group. C a p i l l i n ( X I ) , a conjugated a c e t y l e n i c ketone from Artemesia c a p i l l a r i s Thunb., has a n t i f u n g a l and a n t i - i n f l a m m a t o r y p r o p e r t i e s , and i s a c t i v e a g a i n s t dermal mycoses (33-35). Artemesia monosperma D e l i l e , an E g y p t i a n d e s e r t m e d i c i n a l herb, i s n o t a t t a c k e d by i n s e c t s ( 3 6 ) . I t s a e r i a l p o r t i o n s c o n t a i n an a r o m a t i c d i a c e t y l e n e 3-methyl-3-phenyl-1,4p e n t a d i y n e ( X I I ) which has potent i n s e c t i c i d a l e f f e c t s ( 3 7 ) . M a t r i c a r i a e s t e r ( X I I I ) and i t s d e r i v a t i v e s not only have a n t i f e e d a n t e f f e c t s upon phytophagous i n s e c t s ( 3 8 ) , they i n h i b i t s e e d l i n g g e r m i n a t i o n i n v i t r o ( 3 9 ) . S e v e r a l o t h e r a c e t y l e n e s have been r e p o r t e d t o be p h y t o t o x i c (40,41) a l t h o u g h i t remains u n c l e a r

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

17.

Polyacetylene Photoactivity

MARCHANT AND COOPER

245

whether these compounds a r e a c t u a l l y r e l e a s e d i n t o the r h i z o s p h e r e where a l l e l o p a t h i c i n t e r a c t i o n s a r e thought t o occur (5,42). Structure/Function Relationships i n Polyacetylene P h o t o a c t i v i t y P r i o r t o the d i s c o v e r y o f p o l y a c e t y l e n e p h o t o t o x i c i t y , R e i s c h e t a l (43) had i n v e s t i g a t e d the b a c t e r i o s t a t i c and f u n g i s t a t i c e f f e c t s o f a l a r g e number o f s i m p l e s y n t h e t i c a c e t y l e n e s , i n c l u d i n g hydrocarbons, a c i d s , a l c o h o l s , aldehydes and ketones w i t h one o r two t r i p l e bonds, as w e l l as the C13-ene-tetrayne and pentayne-ene compounds (XIV, XV; Table I I I ) . T h e i r f i n d i n g s suggest t h a t Table I I I . P h o t o a c t i v a t e d p o l y a c e t y l e n e s from the Asteraceae

CH=CH-(C=C)-CH=CH-CH 2

4

3

xnr

CH =CH-(C=C) -CH 2

5

3

JS

OH CH-CH=CH-(C = C) -CH = CH-CH-CH 0H 3

3

2

XVJ CH - (C=C)-CH=CH - O 3

3

xsn

TVITT

CI CH - CH=CH-(C = C)-CH=CH - CH - CH 0H 3

3

2

XIX
3

xx

C i 3 - 1 , 1 1 - d i e n e - 3 , 5 , 7 , 9 - t e t r a y n e ( X I V ) , C13-1-ene-3,5,7,9,11pentayne, Bidens, C o r e o p s i s spp.; s a f y n o l ( X V I ) , Carthamus t i n c t o r i a L.; C i 3 - 5 - e n e - 7 , 9 , 1 1 - t r i y n e - f u r a n ( X V I I ) , Chrysanthemum leucanthemum L.; 2-methyl thiophene ( X V I I I ) , Tagetes spp.; C13-3,11-diene5 , 7 , 9 , t r i y n e - 2 - c h l o r o - 1 - o l ( X I X ) , Centaurea r u t h e n i c a Lam.; 1phenylhepta-1,3,5,-triyne (XX) Bidens a l b a L.

a c e t y l e n e s w i t h a r o m a t i c s u b s t i t u e n t s a r e most a c t i v e and t h a t f u n g i c i d a l e f f e c t s i n c r e a s e w i t h the degree o f u n s a t u r a t i o n i n the

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LIGHT-ACTIVATED PESTICIDES

molecule and polarization of the t r i p l e bond, while compounds which are more hydrophilic tend to be bacteriocidal agents. As d i s t i n c t from the mainly a l i p h a t i c acetylenes of the other families, acetylenes produced by the Asteraceae are characterized by highly unsaturated hydrocarbons and c y c l i c , aromatic or heterocyclic groups ( 1 ) . Some of these complex structures are restricted in d i s t r i b u t i o n while compounds such as thiophenes have been found in the majority of tribes, their occurrence seemingly unrelated to other taxonomic characters ( 3 ) . S i g n i f i c a n t l y photoactive polyacetylenes occur only in the Asteraceae. More than two dozen compounds from the Asteraceae have been extensively tested for photoactivity against various b i o l o g i c a l systems. In general, a l i p h a t i c compounds containing fewer than three conjugated acetylenic bonds do not exhibit phototoxic e f f e c t s against yeasts, filamentous fungi, Gram-negative bacteria, nematodes or mosquito and blackfly larvae (16,44-47) although not a l l compounds with three or t r i p l bond photoactive i photoactivity uniformly (XVI) i s active against but IX i s only phototoxic to nematodes ( 4 5 ) . Extracts from Grindelia species were not photoactive or a n t i b i o t i c to Candida albicans (Robin) Berkh. ( 1 9 ) probably because none of the acetylenes isolated from this genus contain more than two t r i p l e bonds ( 1 ) . S i m i l a r l y , an extensive survey of 80 Asteraceae species for UVmediated a c t i v i t y by Camm et a l . (17) reveals that only those which produce furanoacetylenes (Erigeron spp.), thiarubrines and thiophenes (Tagetes. Heliopsis. Rudbeckia spp.), and a l i p h a t i c compounds with four or f i v e conjugated t r i p l e bonds (Arnica. Centaurea spp.) (1) exhibit phototoxicity to C. albicans. The photoactivity of aromatic and highly unsaturated acetylenes from Bidens and Coreopsis species has also been well documented (48, 4 9 ) . A recent report by Arnason et a_l. (46) showed that furanoacetylenes require three conjugated t r i p l e bonds (XVII) for optimal phototoxicity against Aedes aegypti larvae, and that although methyl- and benzyl-substituted derivatives of alphaterthienyl (II) had absorption spectra similar to II, only 2-methylthiophene (XVIII) was more active. In another study, the r e l a t i v e t o x i c i t y of a series of chemically related polyacetylenes was quantitatively evaluated for a c t i v i t y against Saccharomyces cerevisiae and Escherichia c o l i ( 4 7 ) . The organisms exhibited d i f f e r e n t i a l photosensitivity to some of the compounds but, in general, thiophenes were more toxic than aromatic acetylenes and straight chain hydrocarbons were least active except for a chlorinated ene-triyne-ene alcohol (XIX) whose effects were comparable to that of the thiophenes. It is noteworthy that XIX i s not active against mosquito larvae ( 1 6 ) . The relationship between chemical structure, UV absorption and degree of phototoxicity was examined using another series of naturally-occurring and synthetic acetylenes (Table IV). Phenylacetylene (XXI), phenylpropyne (XXII), diphenylacetylene (XXIV), dithiophene (XXVII) and 8-methoxypsoralen (XVIII) were purchased from Aldrich Chemicals. Compounds II, XX, XXIII, XXV and XXVI were synthesized according to published methods (50-52). 8Methoxypsoralen was used as a reference photoactive compound (53) in

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

17.

247

Polyacetylene Photoactivity

MARCHANT AND COOPER

Table IV. N a t u r a l l y o c c u r r i n g and s y n t h e t i c p o l y a c e t y l e n e s and thiophenes t e s t e d f o r p h o t o t o x i c i t y a g a i n s t microorganisms

CH

CH

XXII

3

OC-OC-CH,

XXIII

-C"C-C-C-C=C-CHj

XX

£3~ s

XXIV

~C3

XXV

s

^Vc_c-c_c-^

XXVII

{MM} s

s

s

OCH

P h e n y l a c e t y l e n e ( X X I ) ; phenylpropyne ( X X I I ) , p h e n y l p e n t a d i y n e ( X X I I I ) , p h e n y l h e p t a t r i y n e (XX), d i p h e n y l a c e t y l e n e (XXIV), d i p h e n y l b u t a d i y n e (XXV), a l p h a - b i t h i e n y l (XXVI), d i t h i e n y l b u t a d i y n e ( X X V I I ) , a l p h a t e r t h i e n y l ( I I ) ; 8-methoxypsoralen ( X X V I I I ) , l i n e a r furanocoumarin used as r e f e r e n c e compound.

the m o d i f i e d d i s c b i o a s s a y o f D a n i e l s (54) d e s c r i b e d elsewhere ( 4 9 ) . Two y e a s t s and s i x b a c t e r i a l s p e c i e s were used a s t e s t o r g a n i s m s , i n c l u d i n g D N A - r e p a i r - d e f i c i e n t mutant E. c o l i B ( _ i ) ( r e c + , e x r - , h r c ~ ) which i s extremely s e n s i t i v e t o UV r a d i a t i o n and t o c h e m i c a l a l k y l a t i n g agents (55,56) (Table V). The two Pseudomonas s p e c i e s a r e p h o t o i n s e n s i t i v e and served as c o n t r o l s . A l l m i c r o o r g a n i s m s were p r o v i d e d by P r o f e s s o r G.H.N. Towers, U n i v e r s i t y o f B r i t i s h Columbia. In the s t a n d a r d b i o a s s a y , 10 gg o f each compound i s a d m i n i s t e r e d and a l l o w e d t o d i f f u s e i n t o the agar f o r 30 m i n u t e s , then t e s t p l a t e s a r e i r r a d i a t e d f o r 2 hours w i t h longwave UV lamps (320-400nm) o f 1.6-2.0Wm~2 i n t e n s i t y a t 15cm. C o n t r o l p l a t e s are kept i n the dark. T o x i c i t y i s measured by the s i z e o f t h e i n h i b i t i o n zones i n the c u l t u r e lawns. D i f f u s i o n e f f e c t s i n t h i s s

American Chemical Society, Library 1155

16th

St., N.W.

Washington, O.C.Heitz, 20036 In Light-Activated Pesticides; J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

248

LIGHT-ACTIVATED PESTICIDES

series i s minimal because of the similar molecular weights and polarity characteristics of the compounds. Results are shown in Table VI. In aromatic acetylenes at least two conjugated t r i p l e Table V. Microorganisms

used in phototoxicity assays.

Escherichia c o l i B/r E. c o l i B(s-1) Pseudomonas aeroginosa P. fluorescens Staphylococcus albus Streptococcus faecal is Bacillus s u b t i l is Candida u t i l i s Saccharomyces cerevisiae

8-METHOXY PSORALEN

-

+++ ++

++

-

-

-

+++

++

++

-

-

+++ +++ +++ ++

-

-

++++ ++++ ++++ ++++

_

_

_

+++

+

++

+++ ++

-

-

++++

+

+++ +++

-

-

++

S. cerevisiae

-

E.coli B/r

Test C o m p o u n d s

C.utilis

+++

T

s

B. subtilis

P. fluorescens

++

Organisms

S. albus

OQ O O uJ

P. aeroginosa

Table VI. Phototoxicit microorganism

+++

Q-CiCH

^ - C i C - C i C - Q

+

+++ ++

Q-CSC-CH3 @-C5C-C3C-CH

3

@-C=C-CsC-C=C-CH

3

++++ +++

++

^-C5C-C5C^

++++ ++++

+

++++

/A

Diameters of clear zones: + 8-12mm.; ++ 12-l8mm.; +++ l8-30mm.; ++++ > 30mm.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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MARCHANT AND COOPER

Polyacetylene Photoactivity

249

bonds are necessary f o r p h o t o a c t i v i t y . An i n s p e c t i o n o f the d a t a i n d i c a t e s t h a t p h o t o a c t i v i t y i s d i r e c t l y c o r r e l a t e d w i t h the number o f a c e t y l e n e s i n the m o l e c u l e . The monoacetylenes do not have s t r o n g a b s o r p t i o n i n the longwave UV r e g i o n ( F i g u r e 4) which suggests poor e f f i c i e n c y o f photon c a p t u r e i n t h a t range. Among the t h i o p h e n e s , II i s more potent than XXVI but not as e f f e c t i v e as XXVII, and d i t h i e n y l b u t a d i y n e (XXVII) i s more t o x i c than d i p h e n y l b u t a d i y n e (XXV), which s u p p o r t s o t h e r o b s e r v a t i o n s t h a t the thiophene moiety has some p a r t i c u l a r l y e f f e c t i v e b i o c i d a l c h a r a c t e r i s t i c s ( 5 , 4 4 ) . The spectrum o f XXVII has a s u b s t a n t i a l longwave component (about 360nm) whereas t h a t o f XXV does not extend much beyond 320nm ( F i g u r e 4 ) . I f p h o t o t o x i c i t y depends on the number o f photons absorbed by the s e n s i t i z i n g molecule and the UV a b s o r p t i o n o f a molecule does not correspond to the emmission spectrum o f the longwave UV s o u r c e , i t would not be expected to have s i g n i f i c a n t p h o t o t o x i c p r o p e r t i e s . McLachlan et a l . (47) have r e p o r t e d t h a t t h i s i s not always t r u e Some p o l y a c e t y l e n e s (XIX and XX) have r e l a t i v e l y lo range y e t are comparabl compounds w i t h s p e c t r a s i m i l a r t o II have no t o x i c i t y a t a l l . It is obvious t h a t _in v i t r o s t u d i e s have t h e i r l i m i t a t i o n s and o t h e r f a c t o r s , not e a s i l y measured, must be important i n such complex biological interactions. P o l y a c e t y l e n e S t r u c t u r e and Mechanisms o f A c t i o n U n l i k e the l i n e a r furanocoumarins, e.g. 8-methoxypsoralen ( X X V I I I ) , which k i l l c e l l s by a photoinduced m o d i f i c a t i o n o f DNA ( 5 7 ) , p h o t o a c t i v e p o l y a c e t y l e n e s and thiophenes a t t a c k c e l l membranes (29,58-61) by photodynamic as w e l l as oxygen-independent mechanisms (62-68). In g e n e r a l , s t r a i g h t c h a i n a l i p h a t i c a c e t y l e n e s such as XIV, XV and XIX, which are n o t o r i o u s l y u n s t a b l e i n v i t r o , have a n o n - o x i d a t i v e mode o f a c t i o n which probably i n v o l v e s the f o r m a t i o n o f f r e e r a d i c a l s upon p h o t o e x c i t a t i o n (62,66). Thiophenes, however, a r e Type I I photodynamic p h o t o s e n s i t i z e r s which damage membranes v i a the c a t a l y t i c g e n e r a t i o n o f s i n g l e t oxygen (58,63,64,66). Partly c y c l i z e d a r o m a t i c a c e t y l e n e s such as p h e n y l h e p t a t r i y n e (XX) which a r e i n t e r m e d i a t e i n s t r u c t u r e between the a l i p h a t i c compounds and the thiophenes a p p a r e n t l y e x h i b i t both photodynamic and nonphotodynamic p r o c e s s e s (66,67). Most a c e t y l e n e s a r e a b l e t o produce s i n g l e t oxygen i n v i t r o a t l e v e l s which do not f u l l y account f o r t h e i r p h o t o t o x i c e f f e c t s , and i n oxygen removal e x p e r i m e n t s , p h e n y l a c e t y l e n e s showed o n l y p a r t i a l or no decrease i n p h o t o t o x i c i t y to microorganisms (66) o r photohemolysis o f e r y t h r o c y t e s ( 2 9 ) . In a d d i t i o n t o the s t a n d a r d p h o t o t o x i c i t y assay which i s done under a e r o b i c c o n d i t i o n s (49,53), the p o l y a c e t y l e n e s e r i e s shown i n T a b l e IV was t e s t e d a g a i n s t seven b a c t e r i a and S. c e r e v i s i a e i n the absence o f oxygen. M a t e r i a l s and the p r o t o c o l d e s c r i b e d f o r BBL GasPak Anaerobic Systems were o b t a i n e d from BBL M i c r o b i o l o g y Systems, P.O.Box 243, C o c k e y s v i l l e MD 21030. Procedures f o r the a n a e r o b i c a s s a y s were i d e n t i c a l t o t h a t d e s c r i b e d above except t h a t s t e p s were taken t o ensure the v i r t u a l absence o f oxygen from the e x p e r i m e n t a l system. A l l media were prereduced i n s e a l e d j a r s and a l l t e c h n i c a l m a n i p u l a t i o n s were c a r r i e d out i n a bag f l u s h e d

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

250

LIGHT-ACTIVATED PESTICIDES

0.4

200

250

300

350

400

F i g u r e 4. U V - a b s o r p t i o n s p e c t r a o f d i p h e n y l a c e t y l e n e (XXIV), d i p h e n y l b u t a d i y n e ( X X V I I ) , and d i t h i e n y l b u t a d i y n e (XXVII).

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Polyacetylene Photoactivity

17. MARCHANT AND COOPER

251

c o n t i n u o u s l y w i t h n i t r o g e n gas. A f t e r the p l a t e s were s t r e a k e d and the compounds a p p l i e d , they were s e a l e d i n a n a e r o b i c pouches t r a n s p a r e n t t o UV and t h e t e s t p l a t e s i r r a d i a t e d . The organisms were then a l l o w e d t o grow a e r o b i c a l l y f o r 24 o r 48 hours. R e s u l t s are shown i n Table V I I and s u p p o r t the d a t a p r e v i o u s l y r e p o r t e d by

8 - M E T H O X Y PSORALEN

+++

+

-

+++

S. cerevisiae

1 .2 B. subtil

S. faeca

(0

S. albus

P. fluonjscens

Test C o m p o u n d s

P. aerocjinosa

E. coli E

Organisms

E. coli E>(s-i)

Table V I I . P h o t o t o x i c i t y o f p o l y a c e t y l e n e s and thiophenes a g a i n s t microorganisms under a n a e r o b i c c o n d i t i o n s

+++

+

++

++

-

+

+++

+

++

Q-CsCH

Ocsc-O Q-C=C-C=CHQ>

-

-

+++

-

-

++++

-

-

+++

Q-CSC-CH3 @ - C s C - C s C - C H

3

@ - C s C - C s C - C s C - C H

3

-

+++

^ - C S C - C S C - ^

++

++++

++++

++

++++ ++

-

++

+++

+++

Diameters o f c l e a r zones: + 8-12mm.; ++ 12-l8mm.; +++ l8-30mm.; ++++ >30mm.

o t h e r s (62-68). The a r o m a t i c a c e t y l e n e s XX, X X I I I and XX were a c t i v e under a e r o b i c and a n a e r o b i c c o n d i t i o n s whereas a l p h a t e r t h i e n y l ( I I ) c l e a r l y r e q u i r e s the presence o f oxygen t o be e f f e c t i v e . Compound XXVII which i s t h e thiophene analogue o f d i p h e n y l b u t a d i y n e (XXV), a l s o o p e r a t e s v i a both types o f mechanisms a l t h o u g h i t i s more t o x i c than t h e l a t t e r .

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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LIGHT-ACTIVATED PESTICIDES

Conclusions P o l y a c e t y l e n e s and thiophenes have both l i g h t - i n d e p e n d e n t and p h o t o a c t i v e e f f e c t s on a wide range o f b i o l o g i c a l systems. There i s a s u b s t a n t i a l body o f evidence which shows t h a t compounds c o n t a i n i n g a t l e a s t two conjugated carbon-carbon t r i p l e bonds can k i l l c e l l s o r i n h i b i t growth by damaging c e l l u l a r membranes. P h o t o a c t i v a t e d a c e t y l e n e s are o f p a r t i c u l a r i n t e r e s t because o f t h e i r p o t e n t i a l a s commercially u s e f u l and e n v i r o n m e n t a l l y n o n - t h r e a t e n i n g b i o c i d a l agents. T h e r e f o r e , the r e l a t i o n s h i p between s t r u c t u r e and f u n c t i o n must be e x p l o r e d , a l t h o u g h many complex and o f t e n unmeasurable f a c t o r s p l a y a r o l e i n b i o l o g i c a l i n t e r a c t i o n s and must be taken i n t o c o n s i d e r a t i o n i n such s t u d i e s . Many d e t a i l s are known about p o l y a c e t y l e n e s and t h e i r i n v i t r o e f f e c t s , but i s must be noted t h a t a l t h o u g h t h e r e i s some c i r c u m s t a n t i a l evidence and c o n s i d e r a b l e s p e c u l a t i o n about t h e i r i n v i v o f u n c t i o n s , t h e r e i s as y e t no c l e a r u n d e r s t a n d i n g o f t h e i r p u t a t i v e r o l e i n nature and a t p r e s e n t no obvious p h y s i o l o g i c a p l a n t s which produce them Acknowledgments T e c h n i c a l a d v i c e f o r the a n a e r o b i c b i o a s s a y s was p r o v i d e d by Derek Ross and h e r e i n g r a t e f u l l y acknowledged. We a l s o thank M e l v i n D.Epp and J . B r i a n Mudd f o r a s s i s t a n c e w i t h the manuscript. Literature 1. 2. 3.

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RECEIVED January21,1987

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

C h a p t e r 18

Thiophenes and Acetylenes: Phototoxic Agents to Herbivorous and Blood-Feeding Insects 1

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J. T. Arnason , B. J . R. Philogène , P. Morand , J . C. Scaiano , N. Werstiuk , and J. Lam 4

1

Ottawa-Carleton Institute for Graduate Studies and Research in Biology and Chemistry, Ottawa K1N 6N5, Canada Division of Chemistry, National Research Council, Ottawa K1A 0R6, Canada Department of Chemistry, McMaster University, Hamilton, Ontario, Canada Department of Organic Chemistry, University of Aarhus, Aarhus, Denmark

2

3

4

Thiophenes and Asteraceae have been found to be highly phototoxic to insects. Effects on herbivorous insects include formation of necrotic lesions, growth reduction and mortality. Mosquito larvae can be controlled at concentrations as low as a few parts per billion. E f f i c i e n t synthetic methods have been developed to produce naturally occurring compounds and derivatives for laboratory and f i e l d trials. A high temperature dilute acid technique has produced t r i t i a t e d phototoxins for pharmacokinetic studies i n i n s e c t s . In fundamental studies on the photos e n s i t i z a t i o n mechanism, laser flash photolysis has been used to determine t r i p l e t lifetimes of the s e n s i t i z e r , rates of energy transfer to O and electron transfer to acceptor, or from donor molecules. The available information suggests that this group of phototoxins has excellent potential for development as i n s e c t i c i d e s . 2

The d i s c o v e r y t h a t many d i v e r s e secondary m e t a b o l i t e s from d i f f e r e n t p l a n t f a m i l i e s a r e p h o t o s e n s i t i z e r s (1,2) suggests n o t o n l y t h a t p h o t o t o x i c i t y has a r i s e n i n d e p e n d e n t l y many times i n e v o l u t i o n as a defense mechanism but t h a t i t may have s i g n i f i c a n t advantages i n d i s c o u r a g i n g p l a n t p e s t s . S t u d i e s o f these naturally occurring systems o f defense p r o v i d e , i n a d d i t i o n , new models f o r t h e development o f p e s t c o n t r o l a g e n t s . The p r e s e n t r e p o r t concerns a l a r g e group o f p h o t o t o x i c compounds, t h e t h i o p h e n e s and b i o s y n t h e t i c a l l y r e l a t e d p o l y a c e t y l e n e s o f t h e A s t e r a c e a e , t h e i r r o l e as a l l e l o c h e r a i c a l s t o i n s e c t p e s t s , and t h e i r p o s s i b l e e x p l o i t a t i o n as i n s e c t i c i d e s . The l i g h t - m e d i a t e d t o x i c i t y t o organisms o t h e r than i n s e c t s by substances from t h i s p l a n t f a m i l y was e s t a b l i s h e d by Gommers' group i n t h e N e t h e r l a n d s (3) and Towers group i n Canada ( 4 ) . S u b s e q u e n t l y , i t was demonstrated t h a t 9 out of 14 compounds t e s t e d were p h o t o t o x i c t o mosquito l a r v a e a t a c o n c e n t r a t i o n o f 0.5 ppm under sources o f 1

0097-6156/87/0339-0255$06.00/0 © 1987 American Chemical Society

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LIGHT-ACTIVATED PESTICIDES near UV i r r a d i a t i o n ( 5 ) . The a c t i v i t y was even g r e a t e r i n s u n l i g h t and two compounds, a l p h a - t e r t h i e n y l (a-T) ( I ) a n d a f u r a n o a c e t y l e n e ( I I ) had e x c e p t i o n a l i n s e c t i c i d a l a c t i v i t y w i t h LC 'S of 19 and 79 ppb r e s p e c t i v e l y ( 6 ) . T h i s u n u s u a l l y h i g h a c t i v i t y l e d t o the p a t e n t i n g of t h e s e substances as i n s e c t i c i d a l agents ( 7 ) and t o the i n v e s t i g a t i o n of the p o t e n t i a l of the compounds as mosquito l a r v i c l d e s . Herbivorous i n s e c t s W h i l e we may h y p o t h e s i z e t h a t p h o t o t o x i c c h e m i c a l s i n the A s t e r a c e a e have a r i s e n i n the course of e v o l u t i o n through s e l e c t i v e p r e s s u r e s e x e r t e d by h e r b i v o r o u s i n s e c t s or p l a n t pathogens, the study of p h o t o s e n s i t l z a t l o n of i n s e c t h e r b i v o r e s has been l i m i t e d because of the l a r g e amount of the c h e m i c a l s r e q u i r e d f o r c a r e f u l i n v e s t i g a t i o n of the e f f e c t of these s u b s t a n c e s on i n s e c t development Downum e t a l (8) and Champagne e t a l . (9) have demonstrate and Euxoa m e s s o r l a induce L a t e r Champagne e t a l . (10) examined the e f f e c t of seven a c e t y l e n e s and t h i o p h e n e s on t h r e e i n s e c t s p e c i e s . S e v e r a l ( I , I I I , I V ) but not a l l of these compounds were p h o t o t o x i c t o M. s e x t a and/or E. m e s s o r l a . However, O s t r i n i a n u b i l a l i s tended t o a v o i d the e f f e c t s of p h o t o s e n s i t i z a t i o n by b u r r o w i n g i n t o d i e t or s p i n n i n g s i l k , b e h a v i o r s which may be a d a p t a t i o n s t o a v o i d p h o t o s e n s i t l z a t l o n by an I n s e c t which i s known t o feed on p h o t o t o x i c A s t e r a c e a e . S p e c i f i c a l l y these t h r e e s t u d i e s i n d i c a t e d t h a t a b s o r p t i o n of l i g h t by the p h o t o t o x i c c h e m i c a l s may i n d u c e m o r t a l i t y , l e n g t h e n l a r v a l development t i m e , and reduce f e e d i n g and growth of s e n s i t i v e i n s e c t s . The most acute e f f e c t s observed were p u p a l d e f o r m i t i e s and n e c r o t i c l e s i o n s i n the c u t i c l e t h a t p r e v e n t e c d y s i s . The e f f e c t s of p h o t o s e n s i t l z a t l o n by a c e t y l e n e s and thiophenes of the A s t e r a c e a e on i n s e c t h e r b i v o r e s i s comparable t o Berenbaum s d e s c r i p t i o n of the e f f e c t s of furanocoumarins of the Apiaceae on unadapted i n s e c t s ( 1 1 ) . I t I s not s u r p r i s i n g t h a t both these f a m i l i e s share an adapted i n s e c t fauna t h a t appears to t o l e r a t e or a v o i d the p h o t o s e n s i t i z i n g chemicals. P h o t o t o x i c a c e t y l e n e s and thiophenes presumably p r o v i d e enhanced p r o t e c t i o n of the p l a n t by v i r t u e of t h e i r i n v o l v e m e n t i n h i g h energy p h o t o c h e m i c a l p r o c e s s e s and the c a t a l y t i c n a t u r e of s i n g l e t oxygen g e n e r a t i o n which they mediate. Thus, p l a n t s may m i n i m i z e t h e i r m e t a b o l i c investment i n c h e m i c a l defenses by a r e l a t i v e l y f r e e commodity, l i g h t , which I s always a v a i l a b l e i n s u i t a b l e p l a n t h a b i t a t s . These compounds are found i n a v a r i e t y of above ground l o c a t i o n s . F o r example, p h e n y l h e p t a t r i y n e (PHT) ( I I I ) i s found i n h i g h c o n c e n t r a t i o n s i n t h e c u t i c l e of l e a v e s of B i d e n s p i l o s a ( 1 ) , w h i l e thiophenes are found i n h i g h c o n c e n t r a t i o n s i n prominent m a r g i n a l l e a f glands of P o r o p h y l l u m spp (Arnason, u n p u b l i s h e d ) . Many members of the s u b t r i b e P e c t i n i d a e are r i c h i n t h i o p h e n e s i n t h e i r above ground parts (38). I t s h o u l d be emphasized, however, t h a t w i t h o u t p h o t o s e n s i t i z i n g r a d i a t i o n , a c e t y l e n e s and thiophenes s t i l l p o s s e s s many of the i n s e c t d e t e r r e n t e f f e c t s observed w i t h o t h e r 1

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n o n - p h o t o s e n s i t i z i n g p l a n t secondary m e t a b o l i t e s e.g. f e e d i n g d e t e r r e n c e , growth r e d u c t i o n , and reduced n u t r i e n t u t i l i z a t i o n (8,9,10,12). The presence of the compounds i n m o d e r a t e l y h i g h c o n c e n t r a t i o n s i n the r o o t s of many A s t e r a c e a e (1) s t i l l suggests a d e f e n s i v e r o l e , a l t h o u g h more e x p e n s i v e m e t a b o l i c a l l y . D i f f e r e n t i a t i o n between l i g h t and dark t o x i c i t y f o r h e r b i v o r o u s i n s e c t s i s not as marked i n microorganisms or mosquito l a r v a e , p o s s i b l y because of the l a c k of l i g h t p e n e t r a t i o n i n t o l a r g e r organisms. Other r e p o r t s of t h e i n s e c t i c i d a l a c t i v i t y of a c e t y l e n e s and t h i o p h e n e s e x i s t i n t h e l i t e r a t u r e (e.g. 12, 14, and 1 5 ) , but l i t t l e a t t e n t i o n was p l a c e d on the p o s s i b l e r o l e of p h o t o s e n s i t l z a t l o n i n these i n v e s t i g a t i o n s . Recent s t u d i e s have attempted t o e x p l a i n why some i n s e c t s p e c i e s a r e more s e n s i t i v e t o a-T than o t h e r s . F o r example, l a t e i n s t a r MU_ s e x t a and P i e r i s rapae a r e v e r y s e n s i t i v e t o a-T but 0. n u b i l a l i s and H e l i o t h l s v l r e s c e n s a r e more t o l e r a n t ( T a b l e 1 ) .

Table 1 Contact P h o t o t o x i c i t y o f q - t e r t h l e n y l to l a s t I n s t a r lavae

Manduca s e x t a P i e r i s rapae H e l i o t h l s vlrescens Ostrinla nubilalis

10 15 474 698

Note: L a r v a e i n t h e i r l a s t i n s t a r were weighed and t r e a t e d w i t h a - t e r t h i e n y l d i s s o l v e d i n acetone a t the f o l l o w i n g r a t e s of a p p l i c a t i o n 0, 1, 3, 30, 100, 300, 1000, ug/g l a r v a e . Insects were i r r a d i a t e d under b l a c k l l g h t b l u e lamps f o r 12 hours a t 2w/m^ and L C v a l u e s c a l c u l a t e d from a p r o b i t a n a l y s i s of the m o r t a l i t y data. 5 Q

I n o r d e r t o e x p l a i n these d i f f e r e n c e s , t r i t i a t e d p h o t o t o x i n s were produced i n h i g h y i e l d (80-100%) by a h i g h temperature d i l u t e a c i d t e c h n i q u e developed by W e r s t i u k (16) f o r m e t a b o l i c s t u d i e s . The procedure was o p t i m i z e d by p r e l i m i n a r y s t u d i e s u s i n g D 0 as the i s o t o p e source b e f o r e f i n a l i n c o r p o r a t i o n of t r i t i u m from HT0. The n a t u r e of the e l e c t r o p h i l i e exchange of a r o m a t i c p r o t o n s p e r m i t s p r e d i c t i o n of t h e r e l a t i v e r a t e s of exchange which has been c o n f i r m e d by NMR s p e c t r o s c o p y . Both H a-T and H-Me-a-T have been produced by t h i s p r o c e d u r e . P h a r m a c o k i n e t i c s t u d i e s (17) have shown t h a t , a f t e r a t o p i c a l a p p l i c a t i o n of t h e l a b e l t o t h e t e s t i n s e c t s , t h e h a l f time f o r c l e a r a n c e was v e r y slow f o r s e n s i t i v e M. s e x t a (48 h r ) , but was much more r a p i d f o r t o l e r a n t (). n u b i l a l i s (6 h r ) and H. v l r e s c e n s (20 h r ) . I n a d d i t i o n , a-T a d m i n i s t e r e d I n t h e d i e t was found t o c r o s s t h e gut and e n t e r the hemolymph t o a g r e a t e r e x t e n t i n M. s e x t a than i n t h e two r e s i s t a n t s p e c i e s . At l e a s t one major 2

3

3

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258

m e t a b o l i t e has been d e t e c t e d i n f e c e s of these i n s e c t s but i t s c h e m i c a l i d e n t i t y i s not y e t known. A p p a r e n t l y , e f f i c i e n t metabolism and c l e a r a n c e of a-T i s one mechanism by which some h e r b i v o r o u s i n s e c t s can d e a l w i t h t h i s p h o t o t o x i c a l l e l o c h e m i c a l . B i o l o g i c a l e f f e c t s at the t h i r d t r o p h i c l e v e l have a l s o been demonstrated w i t h a-T. I n c o r p o r a t i o n of 100 |ig/g of a-T i n t o d i e t s of 0. n u b i l a l i s s i g n i f i c a n t l y reduced the r a t e of p a r a s i t i z a t i o n , s u r v i v a l to p u p a t i o n and a d u l t emergence of the hymenopterous p a r a s i t o i d , Dladegma t e r e b r a n s r e l a t i v e to c o n t r o l s ( 1 8 ) . D e t e c t i o n of a-T i n the a d u l t p a r a s i t o i d suggested t h a t p h o t o s e n s i t l z a t l o n of the p a r a s i t o i d may be p o s s i b l e a l t h o u g h I t has not yet been d i r e c t l y demonstrated. Mosquito l a r v a e The e v a l u a t i o n of a-T or o t h e r m o l e c u l e s as commercial l a r v i c i d e s has been hampered by l a c s y n t h e s e s were e i t h e procedures. A p p l i c a t i o catalyze coupling , r e p o r t e d by Tomao et_ a l . (19) on a m l l l i m o l e s c a l e , has made i t p o s s i b l e to produce a-T on 50 g s c a l e u s i n g r e a d i l y a v a i l a b l e 2-bromothlophene and 2,5-dlbromothlophene as s t a r t i n g m a t e r i a l s . The p r o c e s s i s a one pot Grignard-Wurtz r e a c t i o n I n v o l v i n g a N i c a t a l y s t which d r a s t i c a l l y reduces the f o r m a t i o n of b y p r o d u c t s , f o l l o w e d by a s i m p l e p u r i f i c a t i o n step to g i v e 99% pure m a t e r i a l ( 2 0 ) . A r e c e n t improvement i s the e l i m i n a t i o n of d i e t h y l e t h e r , an i n d u s t r i a l l y u n d e s i r a b l e s o l v e n t , f o r which a p r o c e s s p a t e n t a p p l i c a t i o n (21) has been f i l e d . F i e l d t r i a l s have been u n d e r t a k e n at a deciduous f o r e s t s i t e f o r 3 y e a r s and at a b o r e a l s i t e f o r 2 y e a r s . Spray a p p l i c a t i o n t e s t s of a-T f o r m u l a t e d i n e t h a n o l to n a t u r a l snow melt p o o l s t h a t are Aedes spp. b r e e d i n g s i t e s , r e v e a l e d t h a t e f f e c t i v e c o n t r o l c o u l d be a c h i e v e d at an a p p l i c a t i o n r a t e as low as 10 g a c t i v e i n g r e d i e n t / h a w i t h i n 7 days p o s t a p p l i c a t i o n ( 2 2 ) . More r a p i d (1-2 days p o s t a p p l i c a t i o n ) and r e l i a b l e c o n t r o l i s a c h i e v e d a t lOOg a . i . / h a . T h i s i s comparable to the e f f i c a c y of c u r r e n t l y used organophosphates (e.g. Temephos and P i r i m i p h o s M e t h y l ) but l e s s than t h a t of some p y r e t h r o i d s . Recent work has focussed on the development of a s u i t a b l e c o n c e n t r a t e and an o i l - b a s e d s p r e a d i n g f o r m u l a t i o n t h a t are c o n v e n i e n t to use and are as e f f e c t i v e or b e t t e r than the o r i g i n a l e t h a n o l f o r m u l a t i o n . A p o t e n t i a l l i m i t a t i o n f o r p h o t o t o x i c c o n t r o l agents i s d i m i n i s h e d l i g h t . We have observed reduced e f f i c a c y i n t u r b i d p o o l s as compared to c l e a r p o o l s (Table 2) or under heavy o v e r c a s t as compared to c l e a r , sunny c o n d i t i o n s . However, w i t h i n a s h o r t t i m e , t h e r e i s a comparable t o x i c i t y at the reduced l i g h t l e v e l s u g g e s t i n g t h a t the l i g h t requirement i s s a t u r a t e d f a i r l y rapidly.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

18.

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AL.

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E v a l u a t i o n of e n v i r o n m e n t a l s a f e t y of a-T w i t h r e s p e c t t o n o n - t a r g e t organisms r e q u i r e s more p r e c i s e t o x i c i t y d a t a . This has been a c h i e v e d by l a b o r a t o r y t r i a l s or s i m u l a t e d pond t r i a l s at the f i e l d s i t e w i t h f i e l d c o l l e c t e d mosquitos and n o n - t a r g e t organisms h e l d i n b i o a s s a y cages and p l a c e d i n p l a s t i c wading p o o l s f i l l e d w i t h pond w a t e r . T a r g e t and non t a r g e t data from t h i s t r i a l are shown i n T a b l e 3. A p p l i c a t i o n of 0.10 kg/ha of a-T can e f f e c t i v e l y c o n t r o l mosquito l a r v a e w i t h m i n i m a l e f f e c t s toward damsel and c a d d i s f l y l a r v a e , t r o u t , or s n a i l s . These n o n - t a r g e t r e s u l t s w i t h damsel and c a d d i s f l y are b e t t e r than c u r r e n t l y used p y r e t h r o i d s . However Daphnia and midge are a d v e r s e l y a f f e c t e d at t h i s r a t e of a p p l i c a t i o n by a-T. Table 2 L a r v i c i d a l E f f i c a c y o f q-T Treatment

as a f u n c t i o n o f l i g h t and

time

Day (g/ha)

Turbid pool*

1 2

4365 132

Clear pool

1 2

104 60

* F i n e sediment was d i s t u r b e d i n p o o l s t o c r e a t e a s i t u a t i o n where l i g h t p e n e t r a t i o n of water was reduced. EC f o r mosquito l a r v i c i d e a c t i v i t y was determined as d e s c r i b e d i n ( 2 0 ) . Q

Table 3 T a r g e t and Non-Target Data f o r A l p h a T e r t h i e n y l EC (kg7ha) 5 Q

L C

5n (ppb)

reference

T a r g e t organism mosquito l a r v a e

0.046

15-30

6,22

N o n - t a r g e t organisms damsel f l y l a r v a e 0.38 caddis f l y larvae 1.32 midge l a r v a e 0.178 daphnia l a r v a e 0.044 trout fingerlings 10 snail 10 tadpole 18-111 f a t h e a d minnows 50-650 Note E C v a l u e s are based on s u r f a c e area a p p l i c a t i o n s v a l u e s are c a l c u l a t e d on a c o n c e n t r a t i o n b a s i s . 5 0

22 22 22 22 22 22 23 24 and

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LC

5 0

260

LIGHT-ACTIVATED PESTICIDES Kagan (23-24 and t h i s volume) has r a i s e d e n v i r o n m e n t a l concerns w i t h r e s p e c t to use of a-T as a l a r v i c i d e because of i t s t o x i c i t y to t a d p o l e s and f a t h e a d minnows when f o r m u l a t e d i n d i m e t h y l s u l f o x i d e (DMSO). While the L C v a l u e s f o r these organisms are h i g h e r than f o r mosquito l a r v a e ( T a b l e 3) and t h e use of DMSO as a f o r m u l a t i n g agent may be q u e s t i o n e d , t h e r e i s c l e a r l y a need f o r more n o n - t a r g e t d a t a . E v a l u a t i o n s of the t o x i c o l o g y of a-T show t h a t i t i s n o n - t o x i c to mice at 300 mg/kg by o r a l or dermal r o u t e s of a d m i n i s t r a t i o n s e v e r a l days a f t e r a d m i n i s t r a t i o n ( 2 0 ) , but t h a t i t i s t o x i c to r a t s at 1000 mg/kg i n 24 h r . P h y s i o l o g i c a l e f f e c t s at h i g h doses i n c l u d e CNS d e p r e s s i o n and h y p o t e n s i v e a c t i v i t i e s Te.g. decreased motor a c t i v i t y and l o s s of r i g h t i n g r e f l e x ( 2 5 ) ] . A c e t y l e n e s and thiophenes are non-mutagenic ( 2 6 ) , but can p h o t o s e n s i t i z e human s k i n at h i g h doses ( 1 ) . The l a t t e r was not found to be a problem under d i l u t i o n s used f o r l a r v i c i d i n g . 5 Q

Analogues and d e r i v a t i v e A number of i n v e s t i g a t o r have s y n t h e s i z e d q-T d e r i v a t i v e s and analogues, u s u a l l y by a m u l t i s t e p procedure i n v o l v i n g v a r i o u s c y c l i z a t i o n r e a c t i o n s (see r e f 20). S e v e r a l 1,3-butadiene and thiophene d e r i v a t i v e s prepared i n one of these procedures have been r e p o r t e d to be p h o t o t o x i c t o mosquito l a r v a e on a q u a l i t a t i v e b a s i s ( 2 7 ) . R e c e n t l y , the c a t a l y z e d G r i g n a r d c o u p l i n g r e a c t i o n was used to produce a s e r i e s of compounds (28) s t r u c t u r a l l y r e l a t e d to q-T. LC v a l u e s between 15 and 1000 ppb were r e p o r t e d f o r these compounds. Only one compound, a monomethyl d e r i v a t i v e of a-T ( V ) , was more t o x i c than the parent compound ( T ) . C o n s i d e r a b l e success i n the p e s t i c i d e i n d u s t r y has been a c h i e v e d by the p r o d u c t i o n of designed analogues of n a t u r a l p r o d u c t s such as p y r e t h r i n s and j u v e n i l e hormones. We have i n v e s t i g a t e d the analogues and d e r i v a t i v e s of a-T i n r e l a t i o n to t h e i r p h o t o c h e m i c a l and p h y s i c a l p r o p e r t i e s ( 2 9 ) . L a s e r f l a s h p h o t o l y s i s has demonstrated t h a t most of the compounds have l o n g 5 0

Structures I - V

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

18.

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AL.

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261

t r i p l e t l i f e t i m e s , and t h a t t h e s e t r i p l e t s are e f f i c i e n t l y quenched by 0 to produce s i n g l e t 0 w i t h a quantum y i e l d between 0.4 and 1.0; the quantam y i e l d i s 0.86 f o r a-T i t s e l f which i s one of the most e f f i c i e n t s i n g l e t 0 g e n e r a t o r s i n the s e r i e s t e s t e d . O c t a n o l - w a t e r p a r t i t i o n c o e f f i c e n t s (P) have been e s t i m a t e d to range from l o g P v a l u e s of 4 t o 7. M u l t i p l e r e g r e s s i o n of the d a t a suggested t h a t t o x i c i t y was at l e a s t p a r t i a l l y p r e d i c t a b l e on the b a s i s of p h o t o c h e m i s t r y and p a r t i t i o n c o e f f i c i e n t s . A d e s i g n model based on t h i s and o t h e r i n f o r m a t i o n s h o u l d l e a d to the p r o d u c t i o n of more e f f i c i e n t p h o t o t o x i n s i n the future. 2

2

Mechanisms o f A c t i o n o f q-T, i t s s y n t h e t i c analogues d e r i v a t i v e s , and n a t u r a l l y o c c u r l h g a c e t y l e n e s

and

Assignement of mechanisms of a c t i o n f o r these p h o t o t o x i n s i s c o n t r o v e r s i a l . At th r e p o r t e d to have no 0 Other r e p o r t s have now y single generatio as the i n v i v o and i n v i t r o mode of a c t i o n of q-T ( 1 , 2 ) . S u b s e q u e n t l y , c o n f l i c t i n g i n t e r p r e t a t i o n s have emerged s u g g e s t i n g t h a t p o l y a c e t y l e n e s such as p h e n y l h e p t a t r i y n e (PHT) might a c t by a w h o l l y photodynamic mechanism (31) or by competing photodynamic and f r e e r a d i c a l mechanisms ( 3 2 ) . The r e c e n t a p p l i c a t i o n of l a s e r f l a s h p h o t o l y s i s has r e s o l v e d some of the m e c h a n i s t i c c o n t r o v e r s i e s and p r o v i d e s new i n s i g h t i n t o the p h o t o c h e m i s t r y of these m o l e c u l e s (33-35). With b o t h t h i o p h e n e s and a c e t y l e n e s , l a s e r e x c i t a t i o n r e s u l t s i n the f o r m a t i o n of s t r o n g t r i p l e t s i g n a l s w i t h long l i f e t i m e s (28 us f o r PHT i n MeOH, 30 or 57 us f o r q-T i n MeOH or EtOH r e s p e c t i v e l y ) . The t r i p l e t s are e f f i c i e n t l y quenched by 0 , and by methyl v i o l o g e n but not by amines. W i t h PHT, quenching by 0 and e l e c t r o n t r a n s f e r to m e t h y l v i o l o g e n occur w i t h comparable r a t e c o n s t a n t s ( 3 4 ) , but the back r e a c t i o n of the e l e c t r o n t r a n s f e r (presumably from PHTj" to m e t h y l v i o l o g e n ) was l e s s e f f i c i e n t (50-60%) ( 3 5 ) . R e c e n t l y , d e t e c t i o n of a f r e e r a d i c a l s i g n a l by ESR s p e c t r o s c o p y , i n liposomes c o n t a i n i n g PHT, has been a c h i e v e d a f t e r i r r a d i a t i o n w i t h UV-A ( 3 6 ) . Thus d i r e c t and i n d i r e c t e v i d e n c e suggests t h a t PHT may act by g e n e r a t i o n of f r e e r a d i c a l s or s i n g l e t oxygen. At the b i o c h e m i c a l l e v e l , Kagan (27) has r e p o r t e d I n a c t i v a t i o n of the a c e t y l c h o l i n e s t e r a s e of mosquito l a r v a e under c o n d i t i o n s t h a t induce severe i n s e c t m o r t a l i t y . A more immediate e f f e c t may be the d e s t r u c t i o n of a n a l g i l l membranes of l a r v a e which can be d i r e c t l y observed by l i g h t m i c r o s c o p y . Loss of membrane i n t e g r i t y r e s u l t s i n the r e l e a s e of e l e c t r o l y t e s i n t o w a t e r , and t h i s i s g r e a t l y enhanced under p h o t o s e n s i t i z i n g c o n d i t i o n s (Table 4) at s u b l e t h a l c o n d i t i o n s . W i t h h e r b i v o r o u s i n s e c t s , t h e r e i s e v i d e n c e of i n t e r f e r e n c e w i t h raelanization and s c l e r a t l z a t i o n of pupae (8) and damage to the gut (10) of l a r v a e , i n a d d i t i o n to the d r a m a t i c c u t l c u l a r l e s i o n s . 2

2

2

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262

Table 4 Halide detected

I n démineraitzed Water a f t e r 2 hours (meq/L. ±S.D.) -gT

+gT

-UV

0.080 ±0.012

0.075 ±0.007

+UV

0.105 ±0.004

0.237 ±0.0002

E i g h t y Aedes a t r o p a l p u c o m b i n a t i o n s o f 100 pp deminerallzed water. Halid leakag Chloridometer. No m o r t a l i t y was observed i n t h e 2 h. t r e a t m e n t .

Conclusion R e s i s t a n c e and c r o s s - r e s i s t a n c e a r e major problems w i t h c u r r e n t l y used i n s e c t i c i d e s ( 3 7 ) . The development o f new p e s t c o n t r o l agents i s , f o r t h i s r e a s o n , e s s e n t i a l . With a-T, c r o s s r e s i s t a n c e t o organophosphates does not appear t o be a problem ( 2 4 ) . R e s i s t a n c e t o p h o t o t o x i n s may be slow t o develop i n a q u a t i c l a r v a e because of the r a p i d and n o v e l mode o f a c t i o n o f these s u b s t r a c t s . F i n a l commercial development o f thiophenes o r a c e t y l e n e s w i l l depend, however, on many f a c t o r s i n c l u d i n g p r o p r i e t a r y r i g h t s , market s i z e , and s u i t a b i l i t y f o r r e g i s t r a t i o n . T h e i r demonstrated mode o f a c t i o n and e f f i c a c y and the p r e l i m i n a r y d a t a on t o x i c o l o g y and n o n - t a r g e t e f f e c t s suggests t h a t these s u b s t a n c e s have an e x c e l l e n t p o t e n t i a l f o r t a k i n g t h e i r p l a c e a l o n g s i d e of o t h e r p h o t o t o x i c i n s e c t i c i d e s , h e r b i c i d e s and cheraotherapeutic agents (1,2) i n t h e new p h o t o t o x i n t e c h n o l o g y t h a t i s d e v e l o p i n g i n industry. Acknowledgment T h i s work was supported by t h e Department of N a t i o n a l Defence (Canada) and by the N a t u r a l S c i e n c e s and E n g i n e e r i n g Research C o u n c i l o f Canada. The c o n t r i b u t i o n o f many s t u d e n t s and t e c h n i c i a n s t o t h i s work i s g r a t e f u l l y acknowledged. L i t e r a t u r e Cited 1. 2. 3. 4.

Towers, G.H.N. Can. J. Bot. 1984 62 2900-2911 Knox, J.P.; Dodge, A.D. Phytochem. 1985, 24 889-896 Gommers F.J. Nematology 1972, 18 458 Camm, E.L.; Towers G . H . N . ; M i t c h e l l , J.C., Phytochem 1975, 14, 2007-2011

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5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

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Wat C.K.; Prasad, S.K.; Graham, E.A.; Partington, S.; Arnason, T . , Towers, G.H.N.; Lam, J. Biochem. Syst & Ecol. 1981, 9, 59-62 Arnason, T . ; Swain, T . ; Wat, C.K.; Graham, E . A . ; Partington, S.; Towers, G.H.N, and Lam J. Biochem. Syst. & Ecol. 1981, 9, 63-68 Towers, G.H.N.; Arnason J . T . ; Lambert J.D.H.; Wat C.K., Can Pat. 1,173,743 1984 Downum K.R.; Rosenthal, G.A.; Towers, G.H.N. Pest. Biochem Physiol. 1984 22 104-109 Champagne D.E.; Arnason J . T . ; Philogene B.J.R.; Campbell, G.; and McLachlan, D. Experienta 1984, 40 577-578 Champagne, D . E . , Arnason J . T . , Philogène, B.J.R.; Morand P.; Lam J. J. Chem. Ecol. 1986, 12 835-858 Berenbaum, M. Evolution 1983, 37, 163 McLachlan, D.; Arnason, J . T . ; Philogène, B.J.R.; Champagne, D. Experienta 1982 38 1061-1062 Morallo-Rejesus, B. 31-36 Kakajima, S; Kawazu K. Agric. Biol. Chem. 1977, 41 1801-4 Kawazu, K.; Ariwa, M.; Kii, Y. Agric. Biol. Chem. 1977, 41 223-224 Werstiuk, N.H.; Kadai, T. Can J. Chem. 1973, 51 1485 Iyengar, S.; Arnason T . ; Philogène, B.J.R.; Werstiuk, N.; Morand P . , Abstr. Proc. I.U.P.A.C. Sixth Intemat'l Pesticide. Conf. 1986, Ottawa MacDougall, C. MSc. thesis., University of Ottawa, Ottawa, 1986 Tomao, K . ; Kodoma, S.; Nikajima I; Kumada, M.; Mirato, A.; Suzuki, K. Tetrahed. Lett. 1983, 38; 3347-3354 Philogène, B.J.R.; Arnason J . T . , Berg C.W., Duval F.; Champagne D.; Taylor R.G.; Leitch, L . C . ; Morand, P. J.Econ. Ent. 1985, 78 121-126 Morand, P.; MacEachern A.; Leitch, L . C . ; Arnason, J . T . Can. Patent application filed July, 1986 Philogène, B.J.R.; Arnason, J . T . ; Berg C.W.; Duval, F.; Morand, P. J . Chem Ecol. 1986, 12 893-898 Kagan, J.; Kagan, P.A.; Buhse, H.E. J. Chem Ecol. 1984, 10 1115-1122 Kagan, J.; Kagan E.D.; Seigneurie, W. Chemosphere 1986, 15, 49-57 Towers, G.H.N.; Duangto, K.; Arnason J . T . 1986, unpublished results MacRae, W.D., Chan G.F.Q.; Wat C.K.; Towers, G.H.N.; Lam, J. Experientia 1980, 36 1096-1097 Kagan, J.; Beny, J . P . ; Chan G.; Dahwan, S.N.; Jaworski J . A . ; Kagan, E.D.; Kassner, P.D.; Murphy, M.; Rogers, J.A. Insect Sci. Appl. 1983, 4 377-381 Arnason, J . T . ; Philogène B.J.R.; Berg, C.; Mac Eachern, A.; Kaminski, J.; Leitch, L . C . ; Morand, P.; Lam, J . Phytochem. 1986, 25 1609-1611 MacEachern, A.; Scaiano, J.C; Morand, P.; Arnason, J.T., Campos, F . ; Philogène, B.J.R. Abst. Proc. I.U.P.A.C. Sixth Internat'l Pesticide Conf., Ottawa, 1986

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30. 31. 32. 33. 34. 35. 36. 37. 38.

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Kagan, J.; Gabriel, J . R . ; Reed, S.A. Photochem. Photobiol. 1980, 31 465-469 Kagan, J.; Tadena Wlelant, K.; Chan, G.; Shawan, S.N.; Jaworsky, J . Photochem. Photobiol. 1984, 39 465-467 Evans, C; Weir, D.; Scaiano, J.C.; MacEachern, A.; Arnason, J . T . ; Morand, P.; Hollebone, B.; Leitch, L . C . ; Philogène B.J.R. Photochem. Photobiol. 1986, in press. Weir, D.; Scaiano, J.C.; Arnason, J . T . ; Evans, C. Photochem. Photobiol. 1985, 42 223-230 Reyftmann, J . P . ; Kagan J.; Santos, R.; Morlierre, P. Photochem. Photobiol. 1985, 41 1-7 MacRae, D.; Yammamoto, E.; Towers, G.H.N. 1986. Photochem. Photobioll. in press. Kagan, J.; Hasson, M.; Grynspan F. Biochem. Biophys Acta 1984, 802 442-447 Dover, M . J . ; Croft, B.A. Bioscience 1986, 36 78-85 Downum, K.R.; Keil Ecol. 1986, 13, 109-113

RECEIVED March10,1987

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

C h a p t e r 19 Photodynamic

Action

of

Hypericin

1

J.PaulKnox ,Richard I. Samuels, and Alan D. Dodge School of Biological Sciences, University of Bath, Bath, Avon BA2 7AY, United Kingdom

Hypericin, a photodynami certain specie John's worts). Aspects of its photobiology and photochemistry, especially in relation to its ability to generate singlet molecular oxygen,have been investigated. Its phototoxicity, including that towards Manduca sexta larvae, is also discussed.

The quinones p r o v i d e many examples o f n a t u r a l l y o c c u r i n g photodynamic compounds, and h y p e r i c i n , found p r e d o m i n a n t l y i n t h e H y p e r i c a c e a e i s h i s t o r i c a l l y t h e most i m p o r t a n t o f t h e s e (1#2). The p h o t o s e n s i t i z a t i o n o f g r a z i n g a n i m a l s f o l l o w i n g t h e i n g e s t i o n o f c e r t a i n Hypericum s p e c i e s (the St. John's worts) i s due t o t h e p r e s e n c e o f h y p e r i c i n (3,4). This condition, hypericism, m a n i f e s t i n s k i n i r r i t a t i o n and i n f l a m m a t i o n , i s most commonly caused by t h e i n g e s t i o n o f Hypericum p e r f o r a t u m and has been most p r e v a l e n t i n N o r t h A m e r i c a and A u s t r a l i a (1,5). Chemistry The s t r u c t u r e o f h y p e r i c i n and i t s b i o s y n t h e t i c pathway were e l u c i d a t e d by Brockmann (7,8). H y p e r i c i n (4,5,7,4',5',7'hexahydroxy-2,2'-dimethylnaphthodianthrone) i s a h i g h l y condensed quinone and o f t e n o c c u r s i n t h e presence o f c l o s e l y r e l a t e d photodynamic compounds, most. coir>pionly p s e u d o h y p e r i c i n (9).

'Current address: John Innes Institute, Colney Lane, Norwich NR4 7 U H , United Kingdom

0097-6156/87/0339-0265$06.00/0 © 1987 American Chemical Society

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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0

OH

OH

()

HO'

R

Hypericin

H

HO.

-R

0

2 1

2

R =R =CH

\ OH

1

OH

3

Pseudohypericin 1

2

R =CH OH R =CH 2

3

In e t h a n o l , h y p e r i c i n and p s e u d o h y p e r i c i n display identical and d i s t i n c t i v e a b s o r p t i o n s p e c t r a , w i t h a s e r i e s o f a b s o r p t i o n maxima between 500 and 600nm, and marked red f l u o r e s c e n c e (10,11). They can be d i s t i n g u i s h e d by t h e i r s p e c t r a i n a c i d i c e t h a n o l and aqueous a l k a l i (10). Hypericin i s photostable i n both aqueous and o r g a n i c s o l v e n t s . Distribution The l a r g e s t s u r v e y o f t h e d i s t r i b u t i o n o f h y p e r i c i n w i t h i n t h e H y p e r i c a c e a e r e v e a l e d t h a t approx. 60% o f t h e 200 s p e c i e s i n v e s t i g a t e d contained hypericins. These s p e c i e s were c o n c e n t r a t e d i n t h e s e c t i o n s Euhypericum and Campylosporus (12). T h i s study u t i l i s e d a l e a f p r i n t t e c h n i q u e t h a t was unable t o d i s c r i m i n a t e between h y p e r i c i n and p s e u d o h y p e r i c i n . These compounds do d i f f e r i n t h e i r d i s t r i b u t i o n between s p e c i e s . H. p e r f o r a t u m c o n t a i n s both, IL h i r s u t u m o n l y h y p e r i c i n and H. montanum and fL c r i s p u m o n l y p s e u d o h y p e r i c i n (8). Their d i s t r i b u t i o n w i t h i n p l a n t t i s s u e a l s o d i f f e r s w i d e l y among species. In H^ p e r f o r a t u m t h e l e a v e s , stem and f l o w e r s c o n t a i n the h y p e r i c i n s , whereas i n IL h i r s u t u m h y p e r i c i n o c c u r s o n l y i n the m u l t i c e l l u l a r t r i c l u o i e s o f t h e c a l y x (10). In a l l cases the h y p e r i c i n s a r e r e s t r i c t e d t o d i s c r e t e glands. I n t e r e s t i n g l y , t h e h y p e r i c i n m o l e c u l e appears t o have o t h e r d i v e r s e o c c u r r e n c e s i n nature. The most n o t a b l e examples a r e as the chromophore o f t h e p h o t o r e c e p t o r o f S t e n t o r c o e r u l e u s (a b l u e - g r e e n c i l i a t e ) (13) and i n t h e integument o f an A u s t r a l i a n i n s e c t (Nipaecoccus a u r i l a n a t u s ) (14). I n a d d i t i o n , buckwheat (Fagopyrum esculentum) c o n t a i n s f a g o p y r i n , a d e r i v a t i v e o f h y p e r i c i n (9), t h e mould P e n i c i l l i o p s i s c l a v a r i a e f o r m i s c o n t a i n s p e n c i l l i o p s i n which can be o x i d i s e d and i r r a d i a t e d t o form h y p e r i c i n (9) and t h e c i l i a t e B l e p h a r i s i n a c o n t a i n s a pigment which i s a p o s s i b l e polymer o f h y p e r i c i n (5).

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

19.

KNOX ET AL.

Hypericin

Photodynamic Action of Hypericin

267

and s i n g l e t m o l e c u l a r oxygen

E a r l y s t u d i e s on t h e e f f e c t o f h y p e r i c i n on mammals demonstrated that i t s p h o t o s e n s i t i z i n g a c t i o n r e q u i r e d v i s i b l e l i g h t and oxygen, i.e. was photodynamic. The p r o d u c t i o n o f s i n g l e t m o l e c u l a r oxygen by c e r t a i n photodynamic r e a c t i o n s and i t s r o l e as t h e t o x i c s p e c i e s i n p h o t o o x i d a t i v e damage has s i n c e been d e m o n s t r a t e d (15). We have r e c e n t l y i s o l a t e d h y p e r i c i n from JL h i r s u t u m and i n v e s t i g a t e d i t s p o t e n t i a l t o p h o t o g e n e r a t e s i n g l e t oxygen (10). P u r i f i e d h y p e r i c i n was o b s e r v e d t o promote oxygen consumption from aqueous s o l u t i o n s when i r r a d i a t e d i n t h e presence o f i m i d a z o l e (capable o f r e a c t i n g w i t h s i n g l e t oxygen). T h i s p h o t o o x i d a t i o n was promoted i n t h e p r e s e n c e o f d e u t e r i u m o x i d e and d i m i n i s h e d by the a d d i t i o n o f a z i d e i o n s , s u g g e s t i v e o f s i n g l e t oxygen i n v o l v e m e n t . In a f u r t h e r model system, t h e i r r a d i a t i o n of hyperici o f methyl l i n o l e n a t e , m a l o n d i a l d e h y d e . L i n o l e n a t e o x i d a t i o n was reduced when c r o c i n (a water s o l u b l e c a r o t e n o i d c a p a b l e o f the e f f i c i e n t quenching o f s i n g l e t oxygen) was added t o t h e r e a c t i o n m i x t u r e . In t h i s system a c o n c e n t r a t i o n o f h y p e r i c i n g r e a t e r than lOuM was o b s e r v e d t o reduce l i p i d p e r o x i d a t i o n r e l a t i v e t o c o n t r o l s w i t h o u t h y p e r i c i n (unpublished o b s e r v a t i o n ) . T h i s may r e f l e c t the d i r e c t s c a v e n g i n g o f l i p i d r a d i c a l s by h y p e r i c i n . In both o f the above systems t h e use o f f i l t e r s i n d i c a t e d t h a t t h e e f f e c t i v e i r r a d i a t i o n was 500-600nm. These o b s e r v a t i o n s c l e a r l y demonstrate t h e a b i l i t y o f h y p e r i c i n t o promote type I I photodynamic r e a c t i o n s . Hypericin i s thus p o t e n t i a l l y d i s r u p t i v e o f b i o l o g i c a l s y s t e m s i n w h i c h i t i s i r r a d i a t e d i n p r o x i m i t y t o v u l n e r a b l e c e l l u l a r components such as t h e u n s a t u r a t e d l i p i d s o f membranes (2,15). In a d d i t i o n , e v i d e n c e f o r t h e p h o t o g e n e r a t i o n o f s u p e r o x i d e a n i o n s by t h e i r r a d i a t i o n o f h y p e r i c i n i n a r e d u c i n g environment (in t h e p r e s e n c e o f methionine) has been o b t a i n e d i n a system i n v o l v i n g the r e d u c t i o n o f n i t r o b l u e t e t r a z o l i u m (unpublished observations). The e x t e n t t o which type I photodynamic r e a c t i o n s ( i n c l u d i n g t h e g e n e r a t i o n o f s u p e r o x i d e anions) a r e a component o f t h e photodynamic damage s e n s i t i z e d by h y p e r i c i n i s unknown. Phototoxic

action of hypericin

Photodynamic r e a c t i o n s a r e g e n e r a l l y not s p e c i e s s p e c i f i c . A l t h o u g h h y p e r i c i n p o t e n t i a l l y has a wide t o x i c i t y , i t s a c t i o n w i l l be g r e a t l y modulated by v a r i a t i o n s i n i t s s e q u e s t r a t i o n and m e t a b o l i s m among s p e c i e s and w i t h i n t i s s u e s . As y e t t h e p h o t o t o x i c i t y o f h y p e r i c i n has been i n v e s t i g a t e d i n o n l y a few systems. As a l r e a d y s t a t e d the e a r l y i n v e s t i g a t i o n s upon t h e t o x i c i t y o f h y p e r i c i n were conducted due t o t h e p r e v a l e n c e o f h y p e r i c i s m (5). H y p e r i c i n must be i n g e s t e d by mammals t o r e s u l t i n hypericism and, u n l i k e the f u r a n o c o u m a r i n s , does n o t appear t o be

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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absorbed t h r o u g h the o u t e r l a y e r s of the e p i d e r m i s (5). After i n g e s t i o n a n i m a l s r e m a i n s e n s i t i v e t o s u n l i g h t f o r a week o r more. S k i n i r r i t a t i o n and i n f l a m m a t i o n i s most s e v e r e i n r e g i o n s of unpigmented s k i n d e v o i d of h a i r i.e. the mouth, nose and e a r s (5) . As a l r e a d y p o i n t e d out h y p e r i c i n o c c u r s as a component o f the p h o t o r e c e p t o r of c o e r u l e u s and p r e d i s p o s e s t h i s o r g a n i s m t o l e t h a l photodynamic i n j u r y (16). Exogenous h y p e r i c i n promotes t h i s i n j u r y and the use of quenchers i n t h i s system p r o v i d e s e v i d e n c e o f the i n v o l v e m e n t of s i n g l e t oxygen. The p r e c i s e r o l e of h y p e r i c i n i n Hypericum s p e c i e s i s u n c l e a r , a l t h o u g h i t would appear t o be a d e f e n s i v e one. A l t h o u g h t h i s compound can be a s o u r c e o f i r r i t a t i o n t o mammals, i t i s r a r e l y f a t a l and does not appear t o d e t e r g r a z i n g a n i m a l s (5). I t has been suggested t h a t h y p e r i c i n may a c t as a d e t e r r e n t t o phytophagous i n s e c t s (5), a f r e q u e n t l y proposed r o l e f o r photosensitizing plan i s r e p o r t e d t o be p h o t o t o x i l a r v a e (17). We have u t i l i s e d t h i r d i n s t a r l a r v a e o f the t o b a c c o hawkmoth (Manduca s e x t a , L e p i d o p t e r a : Sphingidae) as a model i n s e c t h e r b i v o r e f o r the i n v e s t i g a t i o n of the p h o t o t o x i c i t y of h y p e r i c i n t o w a r d s i n s e c t s . The normal h o s t range o f s e x t a does not i n c l u d e any s p e c i e s o f the H y p e r i c a c e a e . H y p e r i c i n , i s o l a t e d as d e s c r i b e d p r e v i o u s l y (10), was o b s e r v e d t o be p h o t o t o x i c t o M. s e x t a l a r v a e . At the moderate r a d i a n c e l e v e l used i n t h i s study (22 Wm~ , p r o v i d e d by w h i t e f l u o r e s c e n t tubes) the L D was found t o be 16pg/g l a r v a l i n i t i a l fr.wt., w h i c h r e p r e s e n t s approx. a l u g dose t o a t h i r d i n s t a r l a r v a (Table I ) . In t h e s e e x p e r i m e n t s , the h y p e r i c i n was a d m i n i s t e r e d t o l a r v a e on t o b a c c o l e a f d i s c s (7mm diameter) a f t e r l h o f s t a r v a t i o n from an a r t i f i c i a l d i e t , and o b s e r v a t i o n s were made d u r i n g the subsequent c o n t i n u o u s i r r a d i a t i o n f o r up t o 48h. No m o r t a l i t y or any e f f e c t s upon w e i g h t g a i n were o b s e r v e d i n the h y p e r i c i n t r e a t e d but d a r k - m a i n t a i n e d c o n t r o l l a r v a e . Reduced i r r a d i a n c e r e s u l t e d i n d e c r e a s e d m o r t a l i t y , a l t h o u g h a f t e r 48h the s u r v i v i n g l a r v a e a t the l o w e r l i g h t l e v e l s d i s p l a y e d reduced w e i g h t g a i n r e l a t i v e t o dark c o n t r o l s . The m o d u l a t i o n of l i g h t q u a l i t y by a cut o f f f i l t e r ( a l l o w i n g i r r a d i a t i o n o n l y w i t h wavelengths g r e a t e r than 500nm) reduced the m o r t a l i t y r a t e by o n l y 20%, c o n f i r m i n g t h a t a c t i v e wavelengths i n h y p e r i c i n t o x i c i t y a r e g r e a t e r than 500nm (data not shown). I f a f t e r consumption of the h y p e r i c i n t r e a t e d l e a f d i s c s the l a r v a e were m a i n t a i n e d i n darkness on an a r t i f i c i a l d i e t , t h e p h o t o t o x i c e f f e c t upon subsequent i r r a d i a t i o n was r a p i d l y l o s t . M o r t a l i t y was reduced t o 6% i f i r r a d i a t i o n was d e l a y e d f o r 8h a f t e r t r e a t m e n t (Table I ) . I f the l a r v a e were not s u p p l i e d w i t h a r t i f i c i a l d i e t d u r i n g t h i s p e r i o d o f darkness, the p o t e n t i a l f o r a h i g h m o r t a l i t y r a t e upon subsequent i r r a d i a t i o n o f the l a r v a e s u p p l i e d w i t h d i e t was r e t a i n e d . These o b s e r v a t i o n s suggest t h a t h y p e r i c i n may not be r e a d i l y absorbed by the gut but photoactive a t the gut w a l l and r a p i d l y l o s t from the gut by e x c r e t i o n . 2

5 Q

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

19.

Photodynamic Action of Hypericin

K N O X ET AL.

269

T a b l e I. L e t h a l p h o t o t o x i c i t y o f h y p e r i c i n t o w a r d s Manduca s e x t a larvae. H y p e r i c i n a d m i n i s t e r e d on t o b a c c o l e a f d i s c s . M o r t a l i t y m o n i t o r e d a f t e r 48h o f i r r a d i a t i o n . h y p e r i c i n dose (ug/larva) a

irradiation conditions (Wm~ ) 2

time i n darkness between t r e a t m e n t and i r r a d i a t i o n

percentage mortality 3

ihl 0 0.1 0.3 0.6 1.0 1.5 2.5 2.5

22 " " " " " " DAR

0 " " " " "

0 0 6 12 47 89

2.5 2.5

10 4

" "

15 0

2.0 2.0 2.0 2.0

22 " "

0 2 4

81 34 11 6

"

8

a. average i n i t i a l f r e s h w e i g h t (3rd i n s t a r ) = approx. 60mg. b. a t l e a s t 15 i n s e c t s p e r t r e a t m e n t .

Conclusion These p r e l i m i n a r y o b s e r v a t i o n s d e m o n s t r a t e t h a t o r a l l y a d m i n i s t e r e d h y p e r i c i n i s t o x i c t o l a r v a e o f ML s e x t a , a h e r b i v o r e unaccustomed t o h y p e r i c i n c o n t a i n i n g p l a n t s . This t o x i c i t y has an a b s o l u t e l i g h t dependence a t t h e dose l e v e l s used i n t h i s study, w i t h no m o r t a l i t y o r growth r e t a r d a t i o n o b s e r v e d i n dark m a i n t a i n e d c o n t r o l s . In t h i s c a s e a maximum r a d i a n c e o f 22 Wm was used, c o n s i d e r a b l y l e s s than d a y l i g h t . The L D ^ Q c o u l d t h e r e f o r e be reduced i n a n a t u r a l environment. In t h i s study t h e h y p e r i c i n e q u i v a l e n t t o t h a t c o n t a i n e d i n approx. 50 g l a n d s o f I L h i r s u t u m (10) was l e t h a l t o a t h i r d i n s t a r l a r v a . The l e a f t i s s u e o f ! L p e r f o r a t u m c o n t a i n s h y p e r i c i n a t l e v e l s up t o lmg/g dr.wt. (12). V i s i b l e i r r a d i a t i o n (500-600nm) i s r e q u i r e d for hypericin t o x i c i t y contrasting with that of other plant metabolites capable of p h o t o s e n s i t i z i n g sexta larvae. A t h i o p h e n e , 0 C - t e r t h i e n y l , r e q u i r e d UV i r r a d i a t i o n (320-400nm) f o r i t s a c t i o n (18). The p o s s i b i l i t y t h a t h y p e r i c i n a c t s as a d e t e r r e n t t o phytophagous i n s e c t s r e q u i r e s f u r t h e r t o x i c i t y t e s t s and a survey o f i n s e c t s t h a t u t i l i s e Hypericum s p e c i e s . A beetle, Chrysolina

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b r u n s v i c e n s i s , does f e e d upon h i r s u t u m , u s i n g h y p e r i c i n as a f e e d i n g cue (19). Other C h r y s o l i n a s p e c i e s have been used s u c c e s s f u l l y as a means o f b i o l o g i c a l c o n t r o l o f IL p e r f o r a t u m i n A u s t r a l i a (5^ 20). An a r e a o f i g n o r a n c e h i g h l i g h t e d by t h i s p o s s i b l e case o f c o e v o l u t i o n , i s t h e means by which o r g a n i s m s a r e a b l e t o t o l e r a t e photodynamic a c t i o n . Photodynamic damage may be reduced by b e h a v i o u r a l o r p h y s i o l o g i c a l mechanisms. The mechanisms whereby b i o l o g i c a l systems c o u l d p r e v e n t t h e g e n e r a t i o n o f s i n g l e t m o l e c u l a r oxygen o r w i t h s t a n d i t s s p e c i f i c b u t d i s r u p t i v e o x i d a t i o n s would be o f e s p e c i a l i n t e r e s t .

Literature cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Blum, H.F. 'Photodynamic action and diseases caused by light'; Reinhold Publishing Company, New York, 1941. Knox, J . P . ; Dodge Horsley, C.H. J. Pharmcol Pace, N. Amer. J. Physiol , , Giese, A.C. Photochem. Photobiol. Rev. 1980, 5, 229-255. Towers, G.H.N. Prog. Phytochem. 1980, 6, 183-202. Brockmann, H.H. Prog. Organic Chem. 1952, 1, 64-82. Brockmann, H.H. Proc. Chem. Soc., London 1957, p. 304. Thompson, R.H. 'Naturally occurring Quinones'; Academic:London, 1971. Knox, J.P.;Dodge, A.D. Plant Cell Environ. 1985, 8, 1925. Scheibe, G.; Schöntage, A.Chem.Ber.1942, 75, 20192026. Mathis, C.; Ourisson, G. Phytochem. 1963, 2, 157-171. Walker, E.B.; Lee, T.Y.; Song, P.S. Biochim. Biophys. Acta. 1979, 587, 129-144. Cameron, D.W.; Raverty, W.D. Aus. J. Chem. 1976, 29, 1523-1533. Foote, C.S. In 'Free Radicals in Biology'; Pryor, W.A. Ed; Academic:London, 1976; Vol. II, p.85. Yang, K.C.; Prusti, R.K.; Walker, E.B.; Song, P.S.; Watanabe, M. Furuya, M. Photochem. Photobiol. 1986, 43, 305-310. Arnason, T.; Towers, G.H.N., Philogene, B.J.R.; Lambert, J.D.H. In 'Plant Resistance to Insects'; Hedin, P.A. Ed; ACS Symposium Series No. 208, American Chemical Society: Washington, D.C., 1983, pp. 139-51. Downum, K.R.; Rosenthal, G.A.; Towers, G.H.N. Pest. Biochem. Physiol. 1984, 22, 104-109. Rees, C.J.C. Entomol. Exp. Appl. 1969, 12, 565-583. Clare, N.T. 'Photosensitization in diseases of domestic animals' Commonwealth Agric. Bureaux; England, 1952, pp. 14-15. ;

17.

18. 19. 20.

RECEIVED November 20, 1986

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 20

The

Fungal Photosensitizer

Cercosporin

and

Its

Role

in Plant Disease Margaret E. Daub Department of Plant Pathology, North Carolina State University, Raleigh, NC 27695-7616

Cercosporin i by fungal plan Cercosporin produces singlet oxygen and superoxide when irradiated by light, and damages plants by causing a peroxidation of the membrane lipids. This membrane damage leads to a loss in membrane fluidity, leakage of nutrients, and rapid death of the plant cell. Cercosporin is toxic to mice and bacteria in addition to plants, and attempts to select plant cells in vitro for resistance to cercosporin have not been successful. A large number of fungal species, however, are resistant to cercosporin. Carotenoids and the fungal c e l l wall appear to play a c r i t i c a l role in the resistance of fungi to cercosporin. Cercosporin (1) i s a toxin which appears to play an important role i n plant diseases caused by members of the fungal genus Cercospora. Cercospora species i n c i t e diseases on a large number of host species worldwide, including such crops as corn, soybean, sugar beet, peanut, banana, and coffee. Losses from these diseases can be devastating. In 1985 i n North Carolina alone, Cercospora leaf spot of peanuts caused an estimated 5 m i l l i o n d o l l a r loss with an additional 13 m i l l i o n d o l l a r s spent on control measures to combat the disease; these costs represented almost 15% o f the t o t a l crop v a l u e CI). Cercosrx>ra species are a e r i a l pathogens. Spores produced by these organisms germinate on the leaf surface and enter the leaf through the stomata. Fungal mycelium then ramifies through the leaf i n t e r c e l l u l a r spaces, k i l l i n g the c e l l s and causing severe b l i g h t i n g of the leaf tissue. For many years, i t had been observed that high l i g h t i n t e n s i t i e s were required for symptom development on infected plants (2-4). This e f f e c t was so s t r i k i n g that i t actually l e d to the recommendation i n the 1940's that bananas be grown under p a r t i a l shade to control the disease (5). Although these observations suggested that some type of l i g h t - a c t i v a t e d compound was involved i n 0097-6156/87/0339-0271 $06.00/0 © 1987 American Chemical Society

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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272

.0CH 8 XHJCHOHCHJ CH 2 CH0HCH B OH

1

O

'OCH,

Cercosporin

disease development, i t was not u n t i l the 1970's that cercosporin was i d e n t i f i e d and shown to be l i g h t activated (6-8). Cercosporin Cercosporin was f i r s t isolate Cercospora k i k u c h i i , a soybean pathogen (9). In 1957 Kuyama and Tamura independently isolated the compoun~3 from the same fungus, and named i t cercosporin (10). Cercosporin has since been isolated from a large number of Cercospora species (11-16) and Cercospora-infected plants (10,13,16). Its characterization and structure were reported independently by Lousberg and co-workers (6) and Yamazaki and Ogawa (2). Further studies of i t s stereochemistry have been reported by Nasini and co-workers (17). Okubo et a l . showed that cercosporin i s biosynthesized i n the fungus by the polymerization of acetate and malonate v i a the polyketide pathway (18). N u t r i t i o n a l and environmental conditions regulating toxin biosynthesis by the fungus have also been reported (19). Cercosporin's photosensitizing a c t i v i t y was f i r s t demonstrated by Yamazaki and co-workers i n 1975 (8). They showed that cercosporin was toxic to mice and bacteria only when they were exposed to l i g h t . They further demonstrated an oxygen requirement by the photooxygenation of dimethyl fur an, and showed that cercosporin was capable of degrading amino acids. Hie f i r s t report of the photoactivated t o x i c i t y of cercosporin to plants was that of Macri and V i a n e l l o (20). They demonstrated that cercosporin caused ion leakage from corn, potato, and beet tissues only when they were irradiated by l i g h t . This e f f e c t was observable within 15-30 minutes after treatment and required oxygen. They further found that several synthetic antioxidants could p a r t i a l l y i n h i b i t the cercosporin-induced ion leakage. Our studies on the photosensitizing a c t i v i t y of cercosporin have been done with plant suspension cultures, s i n g l e - c e l l e d cultures of undifferentiated heterotrophic callus c e l l s grown i n l i q u i d medium (21). These cultures are ideal for such studies because they grow equally w e l l i n the l i g h t and dark and lack photosynthetic pigments which could i n t e r f e r e with l i g h t absorption. The use of single c e l l cultures also overcomes other problems encountered with whole tissue studies, because the c e l l s can be exposed uniformly to the toxin, and toxin e f f e c t s can be q u a n t i f i e d by counting the number of c e l l s k i l l e d . The suspension cultures were found to be very s e n s i t i v e to cercosporin. For example, at 5 yM

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

20.

DAUB

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cercosporin, a l l c e l l s i n jl 50 ml tobacco or sugar beet suspension culture (approximately 10 cells) were k i l l e d within 4 hours when i r r a d i a t e d with fluorescent l i g h t s at an i n t e n s i t y of 20 joules" m" "sec" (21). At lower cercosporin concentrations, longer incubation times were required, but even at 0.2 yM, a l l c e l l s were dead within 48 hours. By contrast, i n the dark no c e l l death occurred even at cercosporin concentrations of up to 40 M for up to 7 days. As expected, k i l l i n g of c e l l s by cercosporin was d i r e c t l y proportional to l i g h t dose, with increasing i n t e n s i t y compensating for decreasing exposure times. The wavelength of l i g h t was also c r i t i c a l ; an action spectrum of the k i l l i n g of c e l l s by cercosporin was found to be i n close agreement with the absorption spectrum of cercosporin (21). Cercosporin appears to be able to generate both s i n g l e t oxygen and superoxide when i r r a d i a t e d with l i g h t i n v i t r o (22). Cercosporin, i n the presence of l i g h t , oxygen, and the reducing substrate methionine wa dye readily reduced by t h i s reaction, whereas the s i n g l e t oxygen quencher Dabco (1,4 Diazabicyclo octane) had no effect. Cerosporin also reacted with cholesterol in the presence of l i g h t and oxygen to generate the 5ahydroperoxide of cholesterol, demonstrating the production of s i n g l e t oxygen. Dobrowolski and Foote recently determined the quantum y i e l d of s i n g l e t oxygen formation sensitized by cercosporin to be 0.81 + 0.07 (23). These r e s u l t s do not prove that both s i n g l e t oxygen and superoxide play a role i n the k i l l i n g of c e l l s by cercosporin, but several l i n e s of evidence suggest that both may be involved. The k i l l i n g of suspension culture c e l l s by cercosporin could be s i g n i f i c a n t l y inhibited by the addition of two s i n g l e t oxygen quenchers to the c e l l culture medium, Dabco and b i x i n (21) (bixin i s a carotenoid carboxylic acid which has the same isoprenoid chain length as B-carotene, but i s somewhat soluble i n aqueous solutions). In addition, a low l e v e l of resistance to cercosporin was expressed by a tobacco c e l l culture mutant, selected for resistance to paraquat, which has elevated l e v e l s of superoxide dismutase a c t i v i t y (24). Plants regenerated from t h i s mutant showed no symptoms wEen sprayed with a cercosporin solution and showed l e s s ion leakage following cercosporin treatment than normal tobacco t i s s u e (Tanaka, K., Kyoto P r e f e c t u r a l University, personal communication, 1986). 6

T o x i c i t y to Plant C e l l s The most pronounced e f f e c t seen i n cercosporin-treated plant tissues i s damage to c e l l u l a r membranes. Studies on the u l t r a s t r u c t u r e of Cercospora leaf b l i g h t of sugar beets (25) and of cercosporintreated sugar beet leaves (26) showed membrane damage at e a r l y stages after i n f e c t i o n or toxin treatment. Cercosporin also caused bursting of plant protoplasts (27) and leakage of ions and of the vacuolar pigment betalain (20,27)" from treated c e l l s . These e f f e c t s were very rapid, suggesting that cercosporin has a d i r e c t e f f e c t on membranes. Changes i n e l e c t r o l y t e leakage from tobacco and sugar

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beet leaf disks, for example, could be detected within 1-2 minutes a f t e r treatment with cercosporin i n the l i g h t (27). Evidence from several laboratories demonstrates that t h i s membrane damage i s due to peroxidation of the membrane l i p i d s by cercosporin. C a v a l l i n i and co-workers (28) demonstrated peroxidation of c e l l u l a r constituents in v i t r o . They observed the formation of malondialdehyde (a breakdown product of l i p i d hydroperoxides) and 0 consumption by liposomes and pea and r a t l i v e r mitochondria treated with cercosporin. These reactions could be inhibited by the s i n g l e t oxygen quenchers dimethyl fur an and 3carotene, and by several synthetic antioxidants. In this laboratory we have demonstrated l i p i d peroxidation i n vivo (27). High amounts of ethane (another hydroperoxide product) and malondialdehyde, respectively, were released from cercosporin-treated tobacco leaf disks and suspension cultures when they were incubated i n the l i g h t . An analysis of tobacco suspension culture c e l l s before and a f t e r cercosporin treatment showed larg increase i th r a t i f saturated to unsaturate cercosporin-treated c e l l of the unsaturated f a t t y acids. Further, the addition of <*tocopherol to suspension cultures blocked the l i p i d peroxidation (27). S i m i l a r results were obtained by Youngman et a L (30), who demonstrated ethane release from cercospor in-treated V i c i a faba protoplasts. Peroxidation of p u r i f i e d f a t t y acid methyl esters by cercosporin has also been demonstrated (27,30). As would be expected from the above results, cercosporintreatment of c e l l s r e s u l t s i n marked changes in membrane structure. Electron Spin Resonance (ESR) spectroscopy of cercospor in-treated tobacco protoplasts using two s t e r i c acid spin labels showed a marked decrease i n the f l u i d i t y of the membrane as compared to untreated protoplasts (29). Along with t h i s decrease i n membrane f l u i d i t y was an apparent increase i n the membrane phase transformation temperature as measured by the temperature dependence of f a t t y acid spin l a b e l mobility. Changes such as these have been correlated with increases i n the permeability of the membrane (31), and are commonly seen i n membranes damaged by peroxidation due to ozone or chemical oxidizing agents (31-34). Although photosensitizers have many e f f e c t s on c e l l s and we cannot rule out other s i t e s of action, the membrane-damaging a c t i v i t y of cercosporin appears to be a primary mechanism by which cercosporin destroys plant c e l l s . This mode of action i s also consistent with the etiology of Cercospora diseases. Since Cercospora species do not penetrate the c e l l s of their host plants, they need a mechanism for obtaining nutrients from host c e l l s . By breaking down the host c e l l membranes, cercosporin may provide the fungus with the nutrients required for growth and sporulation within the host. 2

Resistance Mechanisms Mice, bacteria, and a l l plants which have been tested are s e n s i t i v e to cercosporin (8,12-13,35), and i t has not been possible to s e l e c t for cercospor i n - r e s i s t a n t c e l l culture mutants by mutagenesis and selection with cercosporin i n tissue cultures (Daub, M.E.,

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unpublished data). The fungus, however, produces high concentrations of cercosporin i n the l i g h t and i s apparently unaffected by i t . Studies with other fungi have indicated that t h i s resistance i s not unique to Cercospora species, but i s shared by a number of other fungi as w e l l (36). Yeasts, both Saccharomyces cerevisiae and the Basidiomycete yeast Sporobolomyces, are r e s i s t a n t to cercosporin. Among mycelial fungi, several plant pathogens i n the Ascomycete and Deuteromycete classes (for example, A l t e r n a r i a , Fusarium, Colletotrichum, and V e r t i c i l l i u m species) are also highly r e s i s t a n t even though there i s no evidence that they produce s i m i l a r toxins. By contrast, i s o l a t e s of Neurospora crassa and several Aspergillus species, although taxonomically related, were s e n s i t i v e to cercosporin as were fungi i n the Oomycete class. The resistance of a number of d i f f e r e n t fungi to cercosporin i s i n t e r e s t i n g considering the generalized t o x i c i t y of singlet oxygen and superoxide. The current emphasi elucidate the basis of fungi to cercosporin. Our approach has been based on our knowledge of the mode of action of cercosporin and on known differences between r e s i s t a n t and s e n s i t i v e fungi. I t i s important to note that Cercospora species, as w e l l as the other fungi, are r e s i s t a n t to externally supplied cercosporin. Thus compartmentalization of the toxin molecule during synthesis, although possible, cannot completely account for cercosporin resistance. D e t o x i f i c a t i o n of the cercosporin molecule by the fungus i s also not a p o s s i b i l i t y f o r c r y s t a l s of cercosporin are found clustered around the hyphae i n culture. F i n a l l y , r e s i s t a n t and s e n s i t i v e fungi show the same spectrum of s e n s i t i v i t y to hematoporhyrin, another s i n g l e t oxygengenerating photosensitizer, suggesting that the r e s i s t a n t fungi are able to tolerate s i n g l e t oxygen. Several factors have been found not to be important i n resistance (Daub, M. E., unpublished data). Yeasts and the higher mycelial fungi are known to have very saturated membranes as compared to those of the s e n s i t i v e Oomycetes (37), but an analysis of f a t t y acid composition of Cercospora nicotianae demonstrated high concentrations of l i n o l e i c acid, which i s sensitive to peroxidation. Also, although a superoxide dismutase-overproducing mutant of tobacco was found to have low resistance to cerosporin, oxidative enzymes do not appear to be important i n fungal resistance. No s i g n i f i c a n t differences were seen i n superoxide dismutase a c t i v i t y i n extracts from Cercospora species as compared to those from the sensitive fungus Phytophthora cinnamomi, and catalase a c t i v i t y was considerably higher in P. cinnamomi. F i n a l l y , l i t t l e difference was seen i n antioxidant a c t i v i t y of organic and aqueous extracts of s e n s i t i v e and r e s i s t a n t fungi. By contrast, the presence of carotenoids and composition of the fungal c e l l wall do appear to be important i n resistance. Carotenoids. Carotenoids are highly e f f e c t i v e quenchers of s i n g l e t oxygen and of t r i p l e t s e n s i t i z e r s , and appear to be the major means of defense of many organisms against photooxidations s e n s i t i z e d by chlorophyll and a number of photosensitizing dyes (38-39). We have found that Cercospora species produce high concentrations of

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carotenoids (36). In C^ nicotianae, 3-carotene accounts for more than 99% of the t o t a l carotenoids present. 3-carotene production peaks early i n the culture cycle at approximately 12 pg/g dry weight, and then drops o f f , at l e a s t as a function of the dry weight increase. By contrast, two cercospor i n - s e n s i t i v e Phytophthora species, P^ cinnamomi and P. p a r a s i t i c a produce about 20 ng carotenoids per gram dry w~eight. So there are large differences between Cercospora species and some of the cercospor i n - s e n s i t i v e fungi i n carotenoid concentrations. Studies with mutants of Neurospora crassa and Phycomyces blakesleeanus which are blocked i n carotenoid biosynthesis support the hypothesis that carotenoids are important i n fungal resistance to cercosporin. These mutants were s i g n i f i c a n t l y more sensitive t o cercosporin than carotenoid-producing isolates (36). Since carotenoid-def i c i e n t mutants are unavailable i n Cercospora, we attempted to obtain evidence for a role of carotenoids i n Cercospora resistance by blocking carotenoi These included the well-characterize diphenylamine (40), the to block carotenoid synthesis i n fungi as well as plants (40), and the hydroxymethyl-glutaryl-coenzyme A reductase i n h i b i t o r , mevinolin (41). In a l l cases, we were unsuccessful i n blocking 3-carotene production i n C^ nicotianae even though we were able to obtain 7090% i n h i b i t i o n of Neurospora crassa carotenoids with the same compounds (36). At this time we cannot explain why these potent carotenoid "inhibitors were e f f e c t i v e i n i n h i b i t i n g carotenoid synthesis i n N^_ crassa, but not C. nicotianae. I t i s not due to lack of uptake of the d i f f e r e n t i n h i b i t o r s because they had comparable growth reducing e f f e c t s on both fungi. It i s interesting to note that the carotenoid-producing s t r a i n of Neurospora crassa, although more resistant to cercosporin than albino isolates, i s considerably more sensitive than C\ nicotianae, and these two fungi produce comparable amounts of carotenoids (36). These results suggest that carotenoids are important i n resistance, but that they are not the only c e l l u l a r component involved. I t i s also possible that carotenoids play a major role i n resistance of Cercospora species, but that the l o c a l i z a t i o n of carotenoids i n the fungal c e l l d i f f e r s between these two fungi. L o c a l i z a t i o n of the carotenoids i s a c r i t i c a l consideration. Plants, which are very s e n s i t i v e to cercosporin, c e r t a i n l y contain carotenoids, but they are l o c a l i z e d i n chloroplasts and other p l a s t i d s and thus would not be e f f e c t i v e i n protecting membranes i n other parts of the c e l l . Fungal C e l l Wall. Another hypothesis that we have investigated i s that the fungal c e l l w a l l protects the plasma membrane and other parts of the c e l l from cercosporin. I t i s possible that Cercospora species either store the toxin i n t e r n a l l y i n an inactive state and activate i t at the time of excretion (or compartmentalize i t u n t i l excretion), and then by some means either prevent the active molecule from re-entering the c e l l s , or quench the singlet oxygen and superoxide produced by the toxin. Since the toxin acts on membranes, the fungal c e l l w a l l would have to serve as the p r o t e c t i v e barrier. The role of the c e l l w a l l i n resistance was tested by

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determining the s e n s i t i v i t y to cercosporin of fungal protoplasts, generated by digesting the c e l l walls with the enzymes c e l l u l a s e , chitinase, 3-glucuronidase, and amylase. Cercospora nicotianae and Neurospora crassa, although d i f f e r i n g s i g n i f i c a n t l y i n their resistance as mycelium, show the same highly sensitive response t o cercosporin as protoplasts. For example, at 10 uM cercosporin (a concentration which i s not toxic to C^ nicotianae mycelium), protoplasts of both species were completely k i l l e d (42; Qwinn, K. D. and Daub, M. E., unpublished data). As the protoplasts started to regenerate c e l l walls, C^ nicotianae protoplasts rapidly regained resistance (40% were r e s i s t a n t by 4 hours after isolation), whereas the N^_ crassa protoplasts remained very sensitive. These differences were not due to a lag i n c e l l wall regeneration by N. crassa protoplasts. Using resistance to osmotic shock as a measure of c e l l wall regeneration, we found that N^_ crassa protoplasts a c t u a l l y regenerate faster; they s t a r t to become osmotically resistant at approximatel hours for C^ nicotiana The components of the c e l l wall that are important i n resistance have not yet been determined. In our i n i t i a l studies we are looking at the regeneration of wall carbohydrates using o p t i c a l brighteners and fluorescein-tagged l e c t i n s which bind to s p e c i f i c carbohydrate residues (Gwinn, K. D. and Daub, M. E., unpublished data). These compounds cause the protoplasts to fluoresce when they bind to them, and thus the presence of various wall components can be determined by fluoresence microscopy. Based on t h i s technique protoplasts of the two species show the same rate of regeneration o f 3-glucans and of mannose, and have no detectable galactose. The binding of l e c t i n s s p e c i f i c for N-acetyl-glucosamine was s i g n i f i c a n t l y delayed i n N^_ crassa protoplasts, suggesting that c h i t i n deposition occurs l a t e r i n this fungus than i n C^ nicotianae. We do not believe that the presence or absence of c h i t i n plays any role i n resistance, for both fungal species have chitin-containing walls. These data do suggest, however, that there are differences in the walls of these two fungi. Further studies are i n progress. It i s important to note that even though protoplasts of C nicotianae and N^ crassa are quite s e n s i t i v e to cercosporin, they are s t i l l more resistant than plant c e l l s . Approximately 50% of freshly isolated protoplasts of both species survive treatment with 1 yM cercosporin, a concentration that i s l e t h a l to plant c e l l s under the same l i g h t regime (Gwinn, K. D. and Daub, M. E., unpublished data). I t may be that the differences i n s e n s i t i v i t y of C. nicotianae and N^ crassa to cercosporin i s due to protection by the Cercospora c e l l wall, but the basic l e v e l of resistance found i n protoplasts of both species must be due to other factors. The most l i k e l y p o s s i b i l i t y i s protection by carotenoids. As stated previously, these two fungal species produce equivalent amounts of carotenoids. I t i s also possible that the f a i r l y saturated membranes of these fungi are more resistant to peroxidation than the highly unsaturated membranes found i n plants. Further studies are needed to elucidate a l l the factors involved in resistance.

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278 Future Perspectives

Cercospora species are a rather unusual group of plant pathogens. Whereas most plant pathogenic fungi have f a i r l y r e s t r i c t e d host ranges, Cercospora species attack a vast number and d i v e r s i t y of hosts. Further, i t has been hard to control these pathogens, due to the d i f f i c u l t y in i d e n t i f y i n g adequate l e v e l s of resistance i n natural populations of host plants. Much of the success of t h i s group of pathogens may be due to their production of the phytotoxin, cercosporin. No plants which show resistance to cercosporin have been i d e n t i f i e d , and i t has not been possible to induce even moderate levels of cercosporin resistance by mutagenesis and s e l e c t i o n of plant c e l l s with cercosporin i n culture. Cercospora species may i n f a c t be successful because they have the a b i l i t y to produce a molecule against which plants cannot defend themselves. Although photoactivated toxins have not been known to play a role i n other plant diseases, compound isolated from species o suggesting that photosensitizatio y importan pathogenesis for some fungi. I t i s hoped that studies on resistance of fungi to cercosporin w i l l be useful i n developing new and better ways of c o n t r o l l i n g diseases caused by Cercospora species and perhaps other plant pathogenic fungi as well. I f i n fact i t i s not possible to induce resistance to the toxin i n host plants, perhaps the problem can be approached from an opposite, but equally e f f e c t i v e angle, that i s , disruption of the resistance mechanisms of the fungus i t s e l f . This could be accomplished by the use of new non-toxic chemical control targeted at disrupting a resistance mechanism rather than k i l l i n g the fungus. A l t e r n a t i v e l y , i t may be possible to s e l e c t i v e l y breed or genetically engineer plants for the production of compounds that disrupt the fungal defense mechanisms. The p o s s i b i l i t y of genetically modifying plants not to r e s i s t pathogens, but to disrupt their virulence mechanisms, i s an i n t r i g u i n g one that holds promise for future disease control strategies. Literature Cited 1.

2. 3. 4.

5. 6. 7.

Main, C. E.; Byrne, S. V. (eds.). 1985 E s t i m a t e s o f Crop Losses i n North Carolina Due to Plant Diseases and Nematodes; Dept. Plant Pathology Special Publication No. 5, North Carolina State University, Raleigh, N.C., 1986; p 34. Calpouzos, L. Ann. Rev. Phytopathol. 1966, 4, 369-90. Calpouzos, L.; Stallknecht, G. F. Phytopathology 1967, 57, 799-800. Meredith, D. S. Banana Leaf Spot Disease (Sigatoka) caused by Mycosphaerella musicola Leach; Commonwealth Mycological Institute:Kew, Surrey, England, 1970. Thorold, C. A. Trop. Agric. T r i n . 1940, 17, 213-14. Lousberg, R.J.J.Ch.; W e i s s , U.; Salemink, C. A.; Arnone, A.; M e r l i n i , L.; Nasini, G. Chem. Commun. 1971, 1971, 1463-64. Yamazaki, S.; Ogawa, T. Agric. B i o l . Chem. 1972, 36, 170718.

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Yamazaki, S.; Okubo, A.; Akiyama, Y.; Fuwa, K. Agric. B i o l . Chem. 1975, 39, 287-88. 9. Deutschmann, F. Phytopathol. Z. 1953, 20, 297-310. 10. Kuyama, S.; Tamura, T. J . Amer. Chem. Soc. 1957, 59, 572526. 11. Assante, G.; Locci, R.; Camarda, L . ; N e r l i n i , L . ; Nasini, G. Phytochemistry 1977, 16, 243-47. 12. Balis, C.; Payne, M. G. Phytopathology 1971, 61, 1477-84. 13. Fajola, A. O. Physiol. Plant Pathol. 1978, 13, 157-64. 14. Lynch, F. J.; Geoghegan, M. J. Trans. B r i t . Mycol. Soc. 1977, 69, 496-98. 15. Mumma, R. O.; Lukezic, F. L.; Kelly, M. G. Phytochemistry 1973, 12, 917-22. 16. Venkataramani, K. Phytopathol. Z. 1976, 58, 379-82. 17. Nasini, G.; M e r l i n i , L.; Andreettii, G. D.; B o c e l l i , G.; Sgarabotto, P. Tetrahedron 1982, 38, 2787-2796. 18. Okubo, A.; Yamazaki S. Fuwa K Agr B i o l Chem 1975 39 1173-75. 19. Lynch, F. J.; Geoghegan, Mycol , 73, 311-327. 20. Macri, F.; Vianello, A. Plant Cell Environ. 1979, 2, 267-71. 21. Daub, M. E. Phytopathology 1982, 72, 370-74. 22. Daub, M.E.; Hangarter, R. P. Plant Physiol. 1983, 73, 85557. 23. Dobrowolski, D. C; Foote, C. S. Angewante Chemie 1983, 95, 729-30. 24. Furusawa, I.; Tanaka, K.; Thanutong, P.; Mizuguchi, A.; Yazaki, M.; Asada, K. Plant Cell Physiol. 1984, 25, 1247-54. 25. Steinkamp, M. P.; Martin, S. S.; Hoefert, L. L . ; Ruppel, E. G. Physiol. Plant Pathol. 1979, 15, 13-26. 26. Steinkamp, M. P.; Martin, S. S.; Hoefert, L. L . ; Ruppel, E. G. Phytopathology 1981, 71, 1272-81. 27. Daub, M. E. Plant Physiol. 1982, 69, 1361-64. 28. C a v a l l i n i , L . ; Bindoli, A.; Macri, F.; Vianello, A. Chem. Biol. Interactions 1979, 28, 139-46. 29. Daub, M. E . ; Briggs, S. P. Plant Physiol. 1983, 71, 763-66. 30. Youngman, R. J.; Schieberle, P.; Schnabl, H.; Grosch, W.; Elstner, E. F. Photobiochem. Photobiophys. 1983, 6, 109-19. 31. Kunimoto, M.; Inoue, K.; Nojima, S. Biochim. Biophys. Acta 1981, 646, 169-78. 32. Chia, L. S.; Thompson, J. E.; Dumbroff, E. B. Plant Physiol. 1981, 67, 415-20. 33. Dobretsov, G. E.; Borschevskaya, T. A.; Petrov, V. A.; Vladimirov, Y. A. FEBS Lett. 1977, 84, 125-28. 34. Pauls, K. P.; Thompson, J. E. Physiol. Plant. 1981, 53, 25562. 35. Lynch, F. J.; Geoghegan, M. J. Trans. B r i t . Mycol. Soc. 1979, 72, 31-37. 36. Daub, M. E.; Payne, G. A. Phytopathology 1985, 75, 1298 (Abstr.) 37. Wassef, M. K. Adv. L i p i d Res. 1977, 15, 159-232. 38. Krinsky, N. I. Pure Appl. Chem. 1979, 51, 649-660. 39. Foote, C. S.; Denny, R. W.; Weaver, L . ; Chang, Y.; Peters, J . Ann. N. Y. Acad. S c i . 1970, 171, 139-145.

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Ruddat, M.; Garber, E. D. In Secondary Metabolism and D i f f e r e n t i a t i o n i n Fungi; Bennett, J. W.; Ciegler, A., Eds.; Marcel Dekkar Inc.: New York, 1983; Chapter 5. Endo, A.; Kuroda, M.; Tanzawa, K. FEBS L e t t . 1976, 72, 323326. Gwinn, K. D.; Daub, M. E. Phytopathology 1985, 75, 1298 (Abstr.). Overeem, J. C.; S i j p e s t e i j n , A. K. Phytochemistry 1967, 6, 99105. Yoshihara, T.; Shimanuki, T.; A r a k i , T.; Sakamura, S. Agric. B i o l . Chem. 1975, 39, 1683-84. Robeson, D.; S t r o b e l , G.; Matsumoto, G. K.; F i s h e r , E. L.; Chen, M. H.; C l a r d y , J . E x p e r i e n t i a 1984, 40, 1248-50.

RECEIVED

November 20, 1986

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 21

Light-Activated Antimicrobial Chemicals from Plants: Their Potential Role in Resistance to Disease-Causing Organisms 1

Kelsey R. Downum and Stan Nemec

2

1

Department of Biological Sciences, Florida International University, Miami, F L 33199 Horticultural Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Orlando, F L 32803 2

A variety of plant which are e f f e c t i v e , light-activated antimicrobials i n v i t r o . The role of such natural products i n s i t u remains unclear, but their potent biocidal a c t i v i t y suggests that they may represent an important biochemical defense against pathogenic organisms. The first part of t h i s chapter is used to examine what is known concerning the t o x i c i t y of furanocoumarin, polyacetylene and pterocarpan phototoxins toward various plant pathogens. Results of recent studies i n which the s u s c e p t i b i l i t y of nine fungal pathogens of the genus Citrus (Family Rutaceae) to leaf extracts and to various coumarins isolated from three Citrus species (C. limettoides, C. macrophylla and C. medica) are discussed i n the l a t t e r section.

I n a r e c e n t volume on p l a n t d i s e a s e , C o w l i n g and H o r s f a l l (1_) compared p l a n t defense w i t h the defense o f a m e d i e v a l c a s t l e . They p o i n t e d out t h a t h i g h e r p l a n t s , l i k e c a s t l e s , are immobile and must be p r e p a r e d t o p r o t e c t themselves from would-be a t t a c k e r s ( i . e . , h e r b i v o r e s and p o t e n t i a l pathogens) whenever c h a l l a n g e d . The f i r s t l i n e o f defense a g a i n s t i n v a d i n g armies f o r c a s t l e d w e l l e r s was an o u t e r c a s t l e w a l l . E x t e r n a l p l a n t s u r f a c e s ( e . g . , the c u t i c l e , e p i d e r m a l c e l l s , b a r k , e t c . ) s e r v e a s i m i l a r f u n c t i o n and g e n e r a l y p r e c l u d e p a t h o g e n i c organisms from g a i n i n g e n t r a n c e t o i n t e r n a l t i s s u e s . A v a r i e t y o f measures w i t h i n c a s t l e s were a l s o i n p l a c e t o defend c a s t l e i n h a b i t a n t s s h o u l d the o u t e r w a l l s be breached. Mazes of rooms and c o r r i d o r s , t r a p doors and doors w i t h s t u r d y l o c k s , as w e l l as the o c c a s i o n a l s e c r e t passage were i n t e n d e d t o impede t h e p r o g r e s s o f i n v a d i n g armies and a l l o w the c a s t l e d e f e n d e r s time t o regroup o r escape. P l a n t s a l s o have e l a b o r a t e i n t e r n a l d e f e n s e s which e f f e c t i v e l y p r e v e n t p e n e t r a t i o n by p o t e n t i a l pathogens. Such d e f e n s e s i n c l u d e a v a r i e t y o f p h y s i c a l b a r r i e r s , e.g., c e l l u l o s i c w a l l s , l i g n i f i e d and s u b e r i z e d t i s s u e s , as w e l l as a d i v e r s e a r r a y of t o x i c b i o c h e m i c a l s . 0097-6156/87/0339-0281 $06.00/0 © 1987 American Chemical Society

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Endogenous a n t i m i c r o b i a l s a r e among t h e most a c t i v e l y s t u d i e d of t h e v a r i o u s b i o c h e m i c a l defenses e v o l v e d by p l a n t s ( 2 - 7 ) . I n t h i s c h a p t e r , we w i l l r e v i e w what i s c u r r e n t l y known about t h e involvement o f s e v e r a l c l a s s e s o f l i g h t - a c t i v a t e d o r " s o l a r - p o w e r e d " a n t i m i c r o b i a l s i n p l a n t r e s i s t a n c e t o d i s e a s e - c a u s i n g organisms. The r e s u l t s o f r e c e n t work w i t h C i t r u s " p h o t o t o x i n s " o r " p h o t o s e n s i t i z e r s " and t h e i r e f f e c t s on f u n g a l pathogens o f t h a t genus a l s o w i l l be d e s c r i b e d . F i n a l l y , p h y t o c h e m i c a l c l a s s e s w h i c h may mediate p h o t o t o x i c a n t i m i c r o b i a l a c t i v i t y , but have y e t t o be examined f o r such b i o l o g i c a l a c t i o n , w i l l be p o i n t e d o u t . P h o t o s e n s i t i z e r s As P l a n t D e f e n s i v e Agents P l a n t m e t a b o l i t e s from a t l e a s t t e n d i f f e r e n t p h y t o c h e m i c a l c l a s s e s a r e c a p a b l e o f l i g h t - e n h a n c e d t o x i c i t y and may be s y n t h e s i z e d by s p e c i e s i n as many as t e n p e r c e n t o f a l l l i v i n g p l a n t f a m i l i e s ( 8 ) . The c h e m i s t r y , d i s t r i b u t i o n many o f these b i o l o g i c a l l s e v e r a l recent reviews (8-11); , r e p o r t pay a t t e n t i o n t o t h e p o t e n t i a l importance o f endogenous p h o t o t o x i n s i n p l a n t defense a g a i n s t d i s e a s e - c a u s i n g organisms. The involvement o f v a r i o u s p h o t o t o x i c a n t i m i c r o b i a l f u r a n o c o u m a r i n s , p o l y a c e t y l e n e s and p t e r o c a r p a n s i n p l a n t r e s i s t a n c e a r e c o n s i d e r e d s e p a r a t e l y below. Furanocoumarins. P s o r a l e n s o r furanocoumarins o c c u r w i d e l y i n t h e p l a n t kingdom and a r e c h a r a c t e r i s t i c c o n s t i t u e n t s i n t h e A p i a c e a e ( U m b e l l i f e r e a e ) , Fabaceae (Leguminosae), Moraceae and Rutaceae among o t h e r f a m i l i e s (12.13). A p p r o x i m a t e l y 120 d i f f e r e n t d e r i v a t i v e s have been i s o l a t e d and i d e n t i f i e d ( 1 3 ) ; many a r e potent p h o t o t o x i n s c a p a b l e o f k i l l i n g o r i n h i b i t i n g t h e growth o f v i r u s e s , b a c t e r i a and f u n g i as w e l l as a f f e c t i n g a broad-spectrum o f h i g h e r organisms ( s e e r e f . 2. f o r r e v i e w ) . P h o t o a c t i v e furanoucoumarins r e q u i r e e x c i t a t i o n by near u l t r a v i o l e t o r UVA wavelengths (320-400 nm) f o r f u l l expression of t h e i r t o x i c a c t i o n , although light-independent a f f e c t s a r e a l s o known ( s e e d i s c u s s i o n by I v i e , t h i s volume). Covalent bonding t o DNA presumably accounts f o r most o f t h e i r p h o t o t o x i c consequences ( 1 4 ) ; however, p h o t o o x i d a t i v e c e l l u l a r damage ( r e s u l t i n g from e x c i t e d oxygen s p e c i e s ) and p h o t o b i n d i n g t o c e l l u l a r p r o t e i n s have a l s o been demonstrated (15-17). Furanocoumarins a r e g e n e r a l l y p r e s e n t i n h e a l t h y p l a n t t i s s u e s (13,18) where they may f u n c t i o n as preformed o r p r e i n f e c t i o n a l a n t i m i c r o b i a l s which i n h i b i t t h e e s t a b l i s h m e n t o f d i s e a s e - c a u s i n g organisms. S u p r i s i n g l y l i t t l e work has been conducted on t h e t o x i c i t y o f p s o r a l e n s toward p l a n t v i r u s e s , phytopathogenic b a c t e r i a o r f u n g i w h i c h might encounter t h e s e m e t a b o l i t e s i n _ s i t u d e s p i t e our u n d e r s t a n d i n g o f t h e i r p h o t o t o x i c i t y toward o t h e r organisms. The b a c t e r i u m A g r o b a c t e r i u m t u m e f a c i a n s and members o f s e v e r a l f u n g a l genera i n c l u d i n g B o t r y t i s , C e r a t o c y s t i s , S c l e r o t i n i a , Stereum and G l e o s p o r i u m a r e among t h e p h y t o p a t h o g e n i c organisms known t o be a f f e c t e d by v a r i o u s furanocoumarins (19-23). The a c c u m u l a t i o n o r enhanced b i o s y n t h e s i s o f furanocoumarins i n p l a n t t i s s u e s and c e l l s u s p e n s i o n c u l t u r e s exposed t o b i o t i c and a b i o t i c s t r e s s e s suggests t h e i r p o t e n t i a l f o r involvement i n p o s t i n f e c t i o n a l p l a n t responses. The c o n c e n t r a t i o n o f x a n t h o t o x i n

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o r 8-methoxypsoralen (8-MOP; I I ) , one o f t h e most f r e q u e n t l y encountered f u r a n o c o u m a r i n p h o t o s e n s i t i z e r s , d r a m a t i c a l l y i n c r e a s e s i n d i s e a s e d c a r r o t t i s s u e (Daucus c a r o t a ) ( 2 4 ) , i n p a r s n i p (Pastinaca s a t i v a ) root d i s c s inoculated with C e r a t o c y s t i s f i m b r i a t a as w e l l as o t h e r nonpathogens o f p a r s n i p (21) and i n c e l e r y (Apium g r a v e o l a n s ) f o l l o w i n g i n f e c t i o n w i t h t h e " p i n k - r o t " fungus S c l e r o t i n i a s c l e r o t i o r u m ( 1 9 ) . The b i o s y n t h e s i s and a c c u m u l a t i o n o f o t h e r p h o t o t o x i c furanocoumarins i n c l u d i n g bergapten o r 5-methoxypsoralen (5-MOP; I ) ( i n c a r r o t and c e l e r y ) and 4 , 5 , 8 - t r i m e t h y l p s o r a l e n ( i n c e l e r y ) a r e a l s o induced f o l l o w i n g i n f e c t i o n by d i s e a s e - c a u s i n g organisms (24,25). C e l l suspension c u l t u r e s o f p a r s e l y ( P e t r o s e l i n u m h o r t e n s e ) exposed t o f u n g a l e l i c i t o r s accumulate 8-MOP i n a s i m i a r response (26,27). 1

P o l y a c e t y l e n e s and T h e i r D e r i v a t i v e s . P o l y a c e t y l e n e s and t h e i r d e r i v a t i v e s r e p r e s e n t one o f t h e l a r g e s t and most e x h a u s t i v e l y studied c l a s s e s of plan s t r u c t u r e s r a n g i n g fro various r i n g s t a b i l i z e d structures are included i n t h i s c l a s s (28). These p h y t o c h e m i c a l s occur p r i m a r i l y among members o f t h e A p i a c e a e ( U m b e l l i f e r a e ) , A r a l i a c e a e , A s t e r a c e a e (Compositae), Campanulaceae, P i t t o s p o r a c e a e , Oleaceae and S a n t a l a c e a e ( 2 8 ) . A t l e a s t two o t h e r f a m i l i e s , t h e Fabaceae (Leguminosae) and t h e S o l a n a c e a e , have members t h a t produce a c e t y l e n e s ; however these m e t a b o l i t e s a r e s y n t h e s i z e d o n l y i n response t o i n f e c t i o n by p a t h o g e n i c organisms (29,30). Many p o l y a c e t y l e n e s a r e potent p h o t o s e n s i t i z e r s w h i c h can e n t e r an e x c i t e d s t a t e f o l l o w i n g a b s o r p t i o n o f l i g h t energy. I n t h i s e x c i t e d s t a t e , such m o l e c u l e s mediate a v a r i e t y o f broad-spectrum p h o t o t o x i c responses ( s e e 9-11 f o r r e v i e w s ) . Two modes o f a c t i o n have been s u g g e s t e d . S t r a i g h t - c h a i n a c e t y l e n i c m o l e c u l e s tend t o i n t e r a c t d i r e c t l y w i t h t a r g e t b i o m o l e c u l e s i n c e l l s through r a d i c a l mechanisms (31,32) w h i l e t h i o p h e n e s , s u l f u r - d e r i v a t i v e s o f v a r i o u s a c e t y l e n i c p r e c u r s o r s , mediate t h e o x i d a t i o n o f a v a r i e t y o f b i o m o l e c u l e s (membrane l i p i d s and p r o t e i n s i n p a r t i c u l a r ) presumably v i a s i n g l e t oxygen g e n e r a t i o n (32-34). These c o n t r a s t i n g mechanisms a p p a r e n t l y compete i n o t h e r r i n g s t a b i l i z e d a c e t y l e n e s ( 3 2 ) . L i g h t - i n d e p e n d e n t t o x i c i t y has been noted f o r a v a r i e t y o f a c e t y l e n i c m e t a b o l i t e s (31,35); however t h e a c t u a l mechanisms i n v o l v e d i n such i n t e r a c t i o n s have n o t been a d a q u a t e l y studied. The i n v o l v e m e n t o f p o l y a c e t y l e n i c m o l e c u l e s i n p l a n t d i s e a s e r e s i s t a n c e has r e c e i v e d c o n s i d e r a b l e a t t e n t i o n . A t l e a s t 15 p h y t o c h e m i c a l s from t h i s c l a s s have presumed r o l e s as p r e - and/or p o s t i n f e c t i o n a l a n t i m i c r o b i a l agents ( T a b l e I ) . S e v e r a l p o l y a c e t y l e n e s and t h i o p h e n e s i s o l a t e d from members o f t h e A s t e r a c e a e may f u n c t i o n as preformed a n t i b i o t i c s . Such a r o l e i s suggested by t h e i r potent p h o t o t o x i c i t y toward phytopathogens (8,41,43) and because they can be i s o l a t e d from h e a l t h y p l a n t t i s s u e s (28,53,54). Other s t u d i e s r e p o r t t h a t t h e b i o s y n t h e s i s o f a c e t y l e n e s i n whole p l a n t s and i n t i s s u e c u l t u r e s can be s t i m u l a t e d by v a r i o u s f a c t o r s (42,43,55) w h i c h may i n d i c a t e t h e i r p o t e n t i a l i n v o l v e m e n t i n p o s t i n f e c t i o n a l d e f e n s i v e responses. Kourany (43) i n v e s t i g a t e d t h e f a t e o f v a r i o u s a c e t y l e n i c t h i o p h e n e s i n Tagetes

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I. 5-Mtthoxypsoraltn; R , « O M t , R• H II. 8-Mtthoxyptoraltn; R-H,

R „ OMt -

III. 5,8-Dimtthoxypsoraltn; R>OMt, R-OMt T a b l e 1.

P l a n t s which c o n t a i n a n t i f u n g a l p o l y a c e t y l e n e s i n d i c a t e d i n disease resistance

P l a n t Source Aegopodium p o d a g r a r i a L. (Apiaceae) Daucus c a r o t a ( A p i a c e a e ) L y c o p e r s i c o n esculentum (Solanaceae)

Heptadeca-1,9-diene-4,6diyne-3-ol ( f a l c a r i n o l ) 29,36-38 Heptadeca-1,9-diene-4,6diyne-3,8-diol ( f a l c a r i n d i o l )

Dendropanax t r i f i d u s Makino (Araliaceae)

16-Hydroxyoctadeca-9,17-diene12,14-diynoic a c i d Octadeca-9,17-diene-12,14-diyn1,16-diol

B i d e n s p i l o s a L. (Asteraceae)

l-Phenylhepta-l,3,5-triyne

Tagetes e r e c t a L. Tagetes p a t u l a L. (Asteraceae)

2,2 :5 ,2"-Terthienyl 5-(4-Hydroxy-l-butenyl)-2, 2 -bithienyl 5-(4-Acetoxy-l-butenyl)-2, 2 -bithienyl 5-(Buten-3-ynyl)-2, 2 -bithienyl

l

39

40,41

t

f

8,42,43

,

f

Lycopersicon

esculentum

Tetradeca-6-ene-l,3-diyne5,8-diol

29,44

Carthamus t i n c t o r i s (Asteraceae)

Trideca-3,1l-diene-5,7,9triyne-l,2-diol (safynol) Trideca-1l-ene-3,5,7,9-tetrayne1,2-diol (dehydrosafynol)

Lens c u l i n a r i s (Fabaceae) Lens n i g r r i c a n s V i c i a f a b a (Fabaceae) + 31 o t h e r V i c i a s p e c i e s

Wyerone Wyerone A c i d Wyerone Epoxide

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45-48

30,49-52

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e r e c t a L. ( t h e a f r i c a n m a r i g o l d ) i n response t o exposure t o s e v e r a l pathogens. I n o c u l a t i o n of s e e d l i n g s w i t h h i g h l y pathogenic s t r a i n s of A l t e r n a r i a t a g e t i c a and F u s a r i u m oxysporum f . s p . r a d i c i s l y c o p e r i s i c i l e d t o a g e n e r a l d e c r e a s e i n t h i o p h e n e l e v e l s compared t o c o n t r o l p l a n t s . I n f e c t i o n of p l a n t s w i t h Fusarium oxysporum v a r . c a l l i s t e p h i r a c e 2, a m o d e r a t e l y v i r u l e n t pathogen, r e s u l t e d i n a c c u m u l a t i o n of a l p h a - t e r t h i e n y l ( I V ) and two b i t h i o p h e n e d e r i v a t i v e s above l e v e l s encountered i n n o n - i n f e c t e d p l a n t s . In a r e l a t e d s p e c i e s , dwarf m a r i g o l d (Tagetes p a t u l a L.) p l a n t s and t i s s u e c u l t u r e s i n f e c t e d or t r a n s f o r m e d w i t h A g r o b a c t e r i u m t u m e f a c i e n s a l s o accumulated t h i o p h e n e s ( 4 2 ) . These s t u d i e s suggest t h a t t h i o p h e n e b i o s y n t h e s i s can be s t i m u l a t e d by m o d e r a t e l y v i r u l e n t pathogens (which may l e a d t o i n c r e a s e d p l a n t r e s i s t a n c e t o i n f e c t i o n ) , but t h a t h i g h l y pathogenic s p e c i e s may a v o i d t h i s p l a n t response by s u p p r e s s i n g the p r o d u c t i o n of these t o x i c b i o c h e m i c a l s . P h e n y l h e p t a t r i y n e (PHT, V ) , a p h o t o t o x i c p o l y a c e t y l e n e which o c c u r s i n h e a l t h y t i s s u e s of B i d e n a preinfectional inhibito d i s c u s s e d above ( 4 1 ) . Recen pilos i n d i c a t e s t h a t s y n t h e s i s a l s o may be s t i m u l a t e d by a f u n g a l c u l t u r e - f i l t r a t e (55). Three d i a c e t y l e n e a l c o h o l s [ f a l c a r i n o l ( V I ) , f a l c a r i n d i o l and t e t r a d e c a - 6 - e n e - l , 3 - d i y n e - 5 , 8 - d i o l ] , two t r i a c e t y l e n e a l c o h o l s [ d e h y d r o s a f y n o l ( V I I ) and s a f y n o l ] and t h r e e f u r a n o a c e t y l e n e s [wyerone ( V I I I ) , wyerone a c i d and wyerone e p o x i d e ] are a l s o i m p o r t a n t a n t i f u n g a l m e t a b o l i t e s i m p l i c a t e d i n induced r e s i s t a n c e responses i n p l a n t s (30,46-48,50,56). F a l c a r i n o l and f a l c a r i n d i o l occur i n h e a l t h y t i s s u e of F a l c a r i a v u l g a r i s ( 2 8 ) , Daucus c a r o t a (37) and Aegopodium p o d a g r a r i a ( 5 7 ) . These m o l e c u l e s a l s o accumulate r a p i d l y i n c a r r o t r o o t t i s s u e f o l l o w i n g w o u n d - i n o c u l a t i o n w i t h B o t r y t i s c i n e r e a (38) and i n tomato i n f e c t e d w i t h C l a d o s p o r i u m fulvum (29). A t h i r d l i n e a r a c e t y l e n e , c i s - t e t r a d e c a - 6 - e n e - l , 3 - d i y n e - 5 , 8 - d i o l , c o - o c u r r s w i t h f a l c a r i n o l and f a l c a r i n d o l i n d i s e a s e d tomato ( 4 4 ) . S a f y n o l and d e h y d r o s a f y n o l , two t r i a c e t y l e n e a l c o h o l s w i t h pronounced a n t i f u n g a l a c t i v i t y , occur i n s a f f l o w e r (Carthamus t i n c t o r i s ) (45-47). These p a r t i c u l a r m e t a b o l i t e s are r a p i d l y b i o s y n t h e s i z e d by t h i s p l a n t i n reponse t o i n f e c t i o n s w i t h a v i r u l e n t s t r a i n of P h y t o p h t h o r a d r e c h s l e r i and an a v i r u l e n t s t r a i n of megasperma v a r . s o j a e . W i t h i n 48 h of i n o c u l a t i o n , the l e v e l s of s a f y n o l and d e h y d r o s a f y n o l may i n c r e a s e by as much as 40 and 1,500 t i m e s , r e s p e c t i v e l y ( 4 8 ) . The r a t e of d e h y d r o s a f y n o l accumulation i s s t a t i s t i c a l l y c o r r e l a t e d w i t h high disease r e s i s t a n c e i n one p a r t i c u l a r b r e e d i n g l i n e ( B i g g s ) of s a f f l o w e r ( 4 8 ) . Wyerone o c c u r s i n h e a l t h y t i s s u e s of the broad bean V i c i a f a b a L. (49) and a c c u m u l a t e s , u s u a l l y i n c o n j u n c t i o n w i t h wyerone e p o x i d e , i n at l e a s t 28 o t h e r s p e c i e s of V i c i a and two s p e c i e s of Lens when c h a l l e n g e d by H e l m i n t h o s p o r i u m carbonum or B o t r y t i s c i n e r e a ( 3 0 ) . Wyerone a c i d c o - o c c u r s w i t h wyerone and wyerone epoxide i n broad bean p l a n t s i n f e c t e d w i t h s p e c i e s of B o t r y t i s ( 5 2 ) . Despite t h e i r s t r u c t u r a l s i m i l a r i t y with other phototoxic a c e t y l e n e s , f a l c a r i n o l and f a l c a r i n d i o l (and c l o s e l y r e l a t e d m e t a b o l i t e s l i k e f a l c a r i n o n e and f a l c a r i n d i o n e ) , a p p a r e n t l y a r e not p h o t o t o x i c (9). Whether the a n t i m i c r o b i a l a c t i v i t y of t e t r a d e c a - 6 - e n e - l , 3 - d i y n e - 5 , 8 - d i o l , s a f y n o l , d e h y d r o s a f y n o l or the

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

286

LIGHT-ACTIVATED PESTICIDES

wyerone d e r i v a t i v e s may r e s u l t from l i g h t - a c t i v a t e d o r l i g h t - i n d e p e n d e n t p r o c e s s e s remains a c r i t i c a l p o i n t t h a t needs t o be e s t a b l i s h e d . Pterocarpans. S e v e r a l p t e r o c a r p a n d e r i v a t i v e s , most o f t e n r e f e r r e d to i n t h e l i t e r a t u r e as i s o f l a v o n o i d p h y t o a l e x i n s , p r e d o m i n a n t l y o c c u r i n members o f t h e Fabaceae (Leguminosae) ( 3 0 ) . Like furanocoumarins and p o l y a c e t y l e n e s , t h e s e p h y t o c h e m i c a l s a r e t o x i c toward a wide range o f b i o l o g i c a l organisms and accumulate i n p l a n t t i s s u e s i n response t o a v a r i e t y o f s t r e s s e s ( p a r t i c u l a r l y i n f e c t i o n by pathgens) (30,58). F o r t h e s e r e a s o n s , c e r t a i n p t e r o c a r p a n s a r e b e l i e v e d t o p l a y an i m p o r t a n t r o l e i n t h e defense o f p r o d u c i n g p l a n t s a g a i n s t p o t e n t i a l d i s e a s e - c a u s i n g organisms. The b i o s y n t h e s i s , e l i c i t a t i o n and b i o l o g i c a l a c t i v i t y o f i s o f l a v o n o i d p h y t o a l e x i n s has been r e v i e w e d q u i t e r e c e n t l y (58) and w i l l be discussed only b r i e f l y here. Considerable i n t e r e s a n t i m i c r o b i a l pterocarpa involvement i n p l a n t defense larg a v a i l a b l e c o n c e r n i n g t h e c e l l u l a r t a r g e t s and modes o f a c t i o n o f t h e s e p l a n t m e t a b o l i t e s ( 5 8 ) . B a c t e r i a l and f u n g a l membranes a r e p a r t i c u l a r l y s u s c e p t i b l e t o the e f f e c t s of i s o f l a v o n o i d p h y t o a l e x i n s ; however, o t h e r c e l l u l a r s i t e s have n o t been r u l e d out as t a r g e t s o f a c t i o n ( 5 8 ) . One i n v e s t i g a t i o n has examined t h e i n v o l v e m e n t o f l i g h t as an a c t i v a t i n g f a c t o r i n p t e r o c a r p a n t o x i c i t y . Bakker et^ al, (59) found t h a t s e v e r a l p t e r o c a r p a n d e r i v a t i v e s i n c l u d i n g g l y c e o l l i n I ( I X ) , p h a s e o l l i n (X) and p i s a t i n ( X I ) as w e l l as 3 , 6 a , 9 - t r i h y d r o x y p t e r o c a r p a n and t u b e r o s i n c a n form f r e e r a d i c a l s i n t h e presence o f UV i r r a d i a t i o n ( w i t h maximum i n t e n s i t y around 305 nm) and t h a t t h e s e f r e e r a d i c a l s a r e most l i k e l y i n v o l v e d i n t h e i n a c t i v a t i o n o f glucose-6-phosphate dehydrogenase a c t i v i t y i i i v i t r o . The e x t e n t t o w h i c h f r e e r a d i c a l f o r m a t i o n may c o n t r i b u t e t o t h e p t e r o c a r p a n t o x i c i t y (and presumably p l a n t d e f e n s e ) i n o t h e r s t u d i e s where t h e e f f e c t o f l i g h t was n o t c o n s i d e r e d i s n o t c l e a r , but c e r t a i n l y w a r r a n t s f u r t h e r attention. Recent I n v e s t i g a t i o n s Despite demonstrations that v a r i o u s phytochemicals a r e potent l i g h t - a c t i v a t e d a n t i m i c r o b i a l s i n v i t r o and t h a t many a l s o accumulate i n response t o i n f e c t i o n by d i s e a s e - c a u s i n g organisms o r other s t r e s s f u l s i t u a t i o n s , there i s l i t t l e d i r e c t evidence l i n k i n g such m o l e c u l e s t o p l a n t defense in_ s i t u . We have been s t u d y i n g t h e r o l e o f endogenous p h o t o s e n s i t i z e r s t o determine t h e i r i n v o l v e m e n t i n t h e r e s i s t a n c e o f C i t r u s s p e c i e s t o d i s e a s e - c a u s i n g organisms s i n c e f i n d i n g t h a t t h e l e a v e s o f many s p e c i e s c o n t a i n p h o t o s e n s i t i z e r s ( 8 ) . Our e f f o r t s thus f a r have c o n c e n t r a t e d on: 1) e s t a b l i s h i n g t h e s u s c e p t i b i l i t y o f v a r i o u s C i t r u s pathogens t o l e a f e x t r a c t s ; 2) i d e n t i f y i n g t h e p h o t o t o x i c p h y t o c h e m i c a l s i n t h e s e l e a f e x t r a c t s ; and 3) d e t e r m i n i n g pathogen s u s c e p t i b i l i t y t o t h e phytochemicals responsible f o r t h i s b i o c i d a l a c t i o n . I n i t i a l l y , we were i n t e r e s t e d i n d e t e r m i n i n g t h e s u s c e p t i b i l i t y of f u n g a l pathogens i s o l a t e d from C i t r u s t o l e a f e x t r a c t s p r e v i o u s l y

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

21.

DOWNUM AND NEMEC

Light-Activated Antimicrobial Chemicals

287

shown t o e l i c i t p h o t o t o x i c a c t i v i t y ( 8 ) . Nine d i s e a s e - c a u s i n g fungi were o b t a i n e d f o r t h e s e s t u d i e s ( T a b l e I I ) i n c l u d i n g v a r i o u s r o o t , f r u i t and/or l e a f pathogens. S u s c e p t i b i l i t y was determined by s c r e e n i n g t h e p a t h o g e n i c organisms a g a i n s t e x t r a c t s o f C i t r u s l i m e t t o i d e s Tan, (sweet l i m e ) , C. m a c r o p h y l l a Wester (alemow) and C. medica L. ( c i t r o n ) u s i n g a d i s c b i o a s s a y . P l a n t e x t r a c t s were p r e p a r e d by homogenizing f r e s h l y c o l l e c t e d l e a f m a t e r i a l (100 g) i n methanol (300 ml) f o l l o w e d by f i l t r a t i o n and c o n c e n t r a t i o n o f t h e e x t r a c t t o a f i n a l volume o f 10 m l . S t e r i l e f i l t e r paper d i s c s were loaded w i t h t h e d i f f e r e n t e x t r a c t s (20 u l ) and a l l o w e d t o d r y . The d i s c s were p l a c e d onto d u p l i c a t e p o t a t o d e x t r o s e agar (PDA) p l a t e s c o n t a i n i n g e i t h e r s p o r e s o r m y c e l i a l fragments o f t h e n i n e p h y t o p a t h o g e n i c organisms and then i n c u b a t e d i n t h e dark f o r 60 min. Ong o f t h e d u p l i c a t e p l a t e s was i r r a d i a t e d f o r 2 h w i t h UVA (2 W m ) w h i l e t h e o t h e r p l a t e was kept i n t h e dark t o m o n i t o r l i g h t - i n d e p e n d e n t a n t i m i c r o b i a l a c t i o n . A l l p l a t e s were subsequently incubated i 25° then s c o r e d f o r zones o discs.

Table I I .

Fungal pathogens o f C i t r u s

Leaf Pathogens Alternaria c i t r i - leafspot C o l l e t o t r i c h u m gleosporides - anthracnose F r u i t Pathogens A l t e r n a r i a c i t r i - black r o t C o l l e t o t r i c h u m gleosporides - anthracnose D i p l o i d i a n a t a l e n s i s - stem-end r o t Geotrichum candidum - sour r o t P e n i c i I l i u m d i g i t a t u m - green mold P e n i c i I l i u m i t a l i c u m - b l u e mold Root Pathogens Fusarium oxysporum - r o o t r o t Fusarium s o l a n i - r o o t r o t Phytophthora p a r a s i t i c a - foot r o t

Four o f t h e f u n g i t e s t e d were q u i t e s e n s i t i v e t o t h e C i t r u s e x t r a c t s i n t h e presence o f UVA, but were u n a f f e c t e d i n i t s absence ( T a b l e I I I ) . Three o f t h e s u s c e p t i b l e pathogens p r i m a r i l y i n f e c t r o o t s ( F . oxysporum, F. s o l a n i and P h y t o p h t h o r a p a r a s i t i c a ) w h i l e the f o u r t h , C o l l e t o t r i c h u m g l e o s p o r i d i e s , i n f e c t s m a i n l y l e a v e s and f r u i t . Other pathogens o f above-ground p l a n t p a r t s , namely A. c i t r i , D. n a t a l e n s i s , G. candidum and P. d i g i t a t u m , s u c c e s s f u l l y r e s i s t e d the l i g h t - a c t i v a t e d a n t i m i c r o b i a l a c t i o n of a l l three e x t r a c t s . P. i t a l i c u m , however, was s l i g h t l y a f f e c t e d by t h e C i t r u s macrophylla e x t r a c t . F i v e coumarin d e r i v a t i v e s were i d e n t i f i e d i n l e a f e x t r a c t s o f C_. l i m e t t o i d e s , C^. m a c r o p h y l l a and C. medica i n c l u d i n g 5 - h y d r o x y p s o r a l e n , 5-methoxypsoralen ( I ) , 5,8-dimethoxypsoralen ( I I I ) , 4-hydroxycoumarin and 7-hydroxycoumarin [8-MOP a l t h o u g h

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

LIGHT-ACTIVATED PESTICIDES

[nurjLirj Q . . . . « . ^ Kt

IV. Alpha-Terthienyl

K

V. Phenylheptatriyne

HgC-CH-CH-C5C-C= C-CHgCH-CH-CCH^CH OH

3

VI. Falcarinol

H0H C-CH-C=C-C = C - C = C - C « C - C H « C H - C H , o

OH

C H - C H £ CH - CH - C-C-C

CH • CH - CO, Me

VIII. Wyerone

IX. Glyceollin I

X . Phaseollin

XI. Pisatin

MeO

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

+

No i n h i b i t i o n . I n h i b i t i o n zones below 10 mm.

C.

44+++

UVA

limettoides Dark

UVA

Dark

UVA

I n h i b i t i o n zones between 11 - 15 mm. Inhibition zones betweem 16 - 20 mm.

Dark

C_. macrophylla

C. medica

on the growth of Citrus pathogens

Inhibition Zones

UVA-Induced i n h i b i t o r y e f f e c t of crude leaf extracts

Alternaria c i t r i Colletotrichum gleosporides D i p l o i d i a natalensis Fusarium oxysporum Fusarium solani Geotrichum candidum P e n i c i l l i u m digitatum P e n i c i l l i u m italicum Phytophthora p a r a s i t i c a

Pathogens

Table I I I .

290

LIGHT-ACTIVATED PESTICIDES

common i n c l o s e l y r e l a t e d genera has n o t been r e p o r t e d i n C i t r u s ( 1 3 ) ] . The p h o t o t o x i c i t i e s o f t h e s e d e r i v a t i v e s were t e s t e d a g a i n s t the n i n e pathogens l i s t e d i n T a b l e I I . The same b i o a s s a y procedures as above were used except t h a t the i n d i v i d u a l c h e m i c a l s ( d i s s o l v e d i n methanol) were a p p l i e d t o the f i l t e r paper d i s c s i n s t e a d o f l e a f e x t r a c t s . Only 5-methoxypsoralen (5-MOP) e l i c i t e d p h o t o t o x i c responses ( T a b l e I V ) . D. n a t a l e n s i s and the two P e n i c i l l i u m s p e c i e s were r e s i s t a n t i n t h e s e In v i t r o b i o a s s a y s . I n g e n e r a l , t h e s e s t u d i e s suggest t h a t f u n g i w h i c h i n f e c t above-ground p l a n t t i s s u e s are more r e s i s t a n t t o p h o t o t o x i c a c t i o n than r o o t pathogens. Such c o n t r a s t i n g responses p r o b a b l y r e f l e c t an e v o l v e d a b i l i t y by c e r t a i n l e a f and f r u i t pathogens t o c i r c u m v e n t the t o x i c a c t i o n o f t h e s e c h e m i c a l s v i a d e t o x i f i c a t i o n o r o t h e r processes. S i n c e UVA i s r a r e l y e x p e r i e n c e d i n the r h i z o s p h e r e , e v o l u t i o n o f such p r o c e s s e s by r o o t pathogens would seem t o be u n n e c e s s a r y . C o l l e t o t r i c h u m g l e o s p o r i d e s appears t o be an exception. U n l i k e the o t h e pathogen f above-ground t i s s u e s t h i fungus was s u s c e p t i b i l e x t r a c t s which s u g g e s t t i s s u e s may be i n f l u e n c e d by the presence o f endogenous photosensitizers. We have i s o l a t e d s e v e r a l o t h e r p h o t o t o x i c coumarins from v a r i o u s C i t r u s s p e c i e s u s i n g s t a n d a r d b i o a s s a y organisms ( i . e . , E. c o l i and Saccharomyces c e r e v i s i a e ) . I n a d d i t i o n t o 5-MOP, t h e c h e m i c a l s 7-methoxycoumarin and 5 - g e r a n o x y p s o r a l e n ( b e r g a m o t t i n ) have a l s o been i d e n t i f i e d . The t o x i c i t y o f t h e s e m e t a b o l i t e s have not y e t been e s t a b l i s h e d u s i n g the C i t r u s pathogens. Once t h i s has been a c c o m p l i s h e d , we i n t e n d t o q u a n t i t a t e the l e v e l s o f endogenous p h o t o s e n s i t i z e r s i n f i e l d and i n greenhouse-grown C i t r u s p l a n t s and e s t a b l i s h whether p l a n t r e s i s t a n c e t o pathogen i n f e c t i o n c a n be correlated with i n s i t u l e v e l s of p a r t i c u l a r photosensitizers. Conclusion We have d i s c u s s e d the a n t i m i c r o b i a l a c t i v i t y o f more than 20 p h y t o c h e m i c a l s ; most are p o t e n t p h o t o t o x i n s . O t h e r s are i n c l u d e d , not because they are demonstrated p h o t o s e n s i t i z e r s , but because they s h a r e common c h e m i c a l c h a r a c t e r i s t i c s w i t h these b i o l o g i c a l l y a c t i v e p l a n t m e t a b o l i t e s , i . e . , e x t e n s i v e a r o m a t i c o r c o n j u g a t e d double and/or t r i p l e bond systems, and may f u n c t i o n s i m i l a r l y i n v i v o . I n a d d i t i o n t o the m o l e c u l e s a l r e a d y d i s c u s s e d , numerous o t h e r p l a n t - d e r i v e d p h o t o s e n s i t i z e r s are known, but t h e i r r o l e i n p l a n t - p a t h o g e n i n t e r a c t i o n s have y e t t o be e s t a b l i s h e d . Included are v a r i o u s acetophenone, extended q u i n o n e , furanochromone and l i g n a n d e r i v a t i v e s as w e l l as s e v e r a l b e t a - c a r b o l i n e , f u r a n o q u i n o l i n e and i s o q u i n o l i n e a l k a l o i d s ( 8 , 9 ) . Other m e t a b o l i t e s w h i c h are i n v o l v e d w i t h p l a n t r e s p o n s e s t o p a t h o g e n i c i n v a s i o n and have s i m i l a r s t r u c t u r a l f e a t u r e s have r e c e n t l y been i s o l a t e d , e.g., naphthofuranones (60) and d i b e n z o f u r a n s ( 6 1 - 6 3 ) , and w a r r a n t f u r t h e r i n v e s t i g a t i o n w i t h regard t o t h e i r p o t e n t i a l phototoxic a c t i o n . Important a r e a s f o r f u t u r e r e s e a r c h t h a t might a i d i n f u r t h e r e l u c i d a t i n g the s i g n i f i c a n c e o f p h o t o s e n s i t i z e r s i n p l a n t d e f e n s e a g a i n s t d i s e a s e - c a u s i n g organisms i n c l u d e among o t h e r s : 1) t o x i c o l o g i c a l s t u d i e s t o determine c e l l u l a r mechanisms o f pathogen

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Inhibitory

+ ++ +++ I I 11

No i n h i b i t i o n . I n h i b i t i o n zones I n h i b i t i o n zones I n h i b i t i o n zones I n h i b i t i o n zones

_

_

Dark

_ _ _

_

UVA

_

_

UVA

7-HC

below 10 ram. between 11 - 15 mm. between 16 - 20 mm. above 21 mm.

-

-

Dark

4-HC

_

_ -

-

_

+

Dark

UVA

5-MOP

+ -H-f IHI +++ _ _ + +

_

UVA

Zones

Dark

UVA

5,8-diMOP

4-HC - 4-hydroxycoumarin 7-HC - 7-hydroxycoumarin 5-HP - 5 - h y d r o x y p s o r a l e n 5-MOP - 5-methoxypsoralen 5,8-MOP - 5 , 8 - d i m e t h o x y p s o r a l e n

-

Dark

5-KP

Inhibition

e f f e c t o f v a r i o u s coumarins and f u r a n o c o u m a r i n s on C i t r u s pathogens

Alternaria c i t r i Colletotrichum gleosporides Diploidia natalensis Fusarium oxysporum Fusarium s o l a n i Penicillium digitatum Penicillium italicum Phytophthora p a r a s i t i c a

Pathogens

T a b l e IV.

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LIGHT-ACTIVATED PESTICIDES

s u s c e p t i b i l i t y ( o r r e s i s t a n c e ) t o p h o t o t o x i c m e t a b o l i t e s ; 2) p h y t o c h e m i c a l s t u d i e s t o e v a l u a t e the q u a n t i t a t i v e v a r i a t i o n o f s p e c i f i c p h o t o t o x i n s i n p l a n t p o p u l a t i o n s coupled w i t h i n s i t u s t u d i e s t h a t attempt t o c o r r e l a t e those endogenous l e v e l s w i t h p l a n t r e s i s t a n c e ( o r s u s c e p t i b i l i t y ) t o s p e c i f i c pathogens; and 3) breeding studies t o s e l e c t f o r plant l i n e s that synthesize a g r a d i e n t o f endogenous p h o t o s e n s i t i z e r c o n c e n t r a t i o n s w h i c h can be e v a l u a t e d f o r r e s i s t a n c e t o a broad range o f v i r u l e n t organisms. I n a d d i t i o n , past s t u d i e s t h a t i n v o l v e d p l a n t p h o t o s e n s i t i z e r s , but d i d not c o n s i d e r l i g h t as an a c t i v a t i n g element i n t h e i r t o x i c i t y , need t o be r e - e v a l u a t e d . Acknowledgments

We would l i k e t o express our g r a t i t u d e t o Dr. Stewart A. Brown ( T r e n t U n i v e r s i t y ) f o r p r o v i d i n g us w i t h s t a n d a r d s o f v a r i o u s f u r a n o c o u a r i n s and L a v i n F a l e i r o A d e l h e i d R e i n o s o Johann S c o t t and Lee Swain f o r t e c h n i c a g r a n t s from the W h i t e h a l

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Cowling, E.B.; H o r s f a l l , J.G. In Plant Disease An Advanced Treatise. How Plants Defend Themselves; H o r s f a l l , J.C.; Cowling, E.B., Eds.; Academic: New York, 1980, Vol. V, pp. 1-16. Overeem, J.C. In Biochemical Aspects of Plant-Parasite Relationships; Friend, J . ; T h r e l f a l l , D.R., Eds.; Academic: New York, 1976, pp. 195-206. Kuc, J . ; Shain, L. In Antifungal Compounds. Interactions in Biological and Ecological Systems; Siegel, M.R.; S i s l e r , H.D., Eds.; Marcel Dekker: New York, 1977, Vol 2, pp. 497-535. Swain, T. Ann. Rev. Plant Physiol. 1977,28,479-501. B e l l , A.A. Ann. Rev. Plant Physiol. 1981,32,21-81. Smith, D.A. In Phytoalexins; Bailey, J.A.; Mansfield, J.W., Eds.; Wiley: New York, 1982, pp. 218-52. D a r v i l l , A.G.; Albersheim, P. Ann. Rev. Plant Physiol. 1984, 35, 243-75. Downum, K.R. In Natural Resistance of Plants to Pests: Roles of Allelochemicals; Green, M.B.; Hedin, P.A., Eds.; ACS SYMPOSIUM SERIES No. 296, American Chemical Society: Washington, D.C., 1986, pp. 197-205. Towers, G.H.N. Can. J . Bot. 1984,62,2900-11. Knox, J.P.; Dodge, A.D. Phytochem. 1985,24,889-96. Downum, K.R.; Rodriguez, E. J . Chem. Ecol. 1986, 12, 823-34. Gray, A.I.; Waterman, P.G. Phytochem. 1978,17,845-64. Murray, R.D.H.; Mendez, J . ; Brown, S.A. The Natural Coumarins: Occurrence, Chemistry and Biochemistry; Wiley: New York, 1982. Song, P.-S.; Tapley, K.J., J r . Photochem. Photobiol. 1979, 29, 1177-97. Veronese, F.M.; Schiavon, O.; Bevilacqua, R.; Bordin, F; Rodighiero, G. Photochem. Photobiol. 1982, 36, 25-30.

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16. 17. 18. 19. 20. 21. 22.

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Granger, M.; Helene, C. Photochem. Photobiol. 1983, 38, 563-68. Tuveson, R.W.; Berenbaum, M.R.; Heininger, E.E. J . Chem. Ecol. 1986, 12, 933-48. Beier, R.C.; I v i e , G.W.; O e r t l i , E.H.; Holt, D.L. Fd. Chem. Toxic. 1983, 21, 163-65. Scheel, L.D.; Perone, V.B.; Larkin, R.L.; Kupel, R.E. Biochem. 1963, 2, 1127-31. Martin, J.T.; Baker, E.A.; Byrde, R.J.W. Ann. Appl. B i o l . 1966, 57, 491-500. Johnson, C.; Brannon, D.R.; Kuc, J . Phytochem. 1973, 12, 2961-62. Martin, J.T. In Fungal Pathogenicity and the Plant's Response; Byrde, R.J.W.; Cutting, C.V., Eds.; Academic: New York, 1973, pp. 333-5. Afek, U.; Sztejnberg, A. Phytochem. 1986, 25, 1855-56. Ceska, O.; Chaudhary, S.K.; Warrington, P.J.; Ashwood-Smith, M.J. Phytochem. 1986 Wu, C.M.; Koehler 23., 852-56. Chappel, J ; Hahlbrock, K. Nature 1984,311,76-78. Hauffe, K.D.; Hahlbrock, K.; Scheel, D. Z. Naturforsch. 1986, 41c, 228-39. Bohlmann, F; Burkhardt, T; Zdero, C. Naturally Occurring Acetylenes; Academic: London, 1973. de Wit, P.J.G.M.; Kodde, E. Physiol. Plant Path. 1981, 18, 143-48. Robeson, D.J.; Harborne, J.B. Phytochem. 1980, 19, 2359-65. McLachlan, D.; Arnason, J.T.; Lam, J . Photochem. Photobiol. 1984, 39, 177-82. McLachlan, D.; Arnason, T.; Lam, J . Biochem. System. Ecol. 1986, 14, 17-23. Arnason, T.; Chan, G.F.Q.; Wat, C.-K.; Downum, K.R.; Towers, G.H.N. Photochem. Photobiol. 1981, 33, 821-24. Downum, K.R.; Hancock, R.E.W.; Towers, G.H.N. Photochem. Photobiol. 1982, 36, 517-23. Champagne, D.E.; Arnason, J.T.; Philogene, B.J.R.; Morand, P.; Lam, J . J . Chem. Ecol. 1986, 12, 835-58. Kemp, M.S. Phytochem. 1978,17,1002. Garrod, B.; Lewis, B.G.; Coxon, D.T. Physiol. Plant Path. 1978, 13, 241-46. Harding, V.K.; Heale, J.B. Physiol. Plant Path. 1980, 17, 277-89. Hansen, L.; B o l l , P.M. Phytochem. 1986, 25, 285-93. DiCosmo, F.; Towers, G.H.N.; Lam, J . Pestic. S c i . 1982, 13, 589-94. Bourque, G.; Arnason, J.T.; Madhosingh, C.; Orr, W. Can. J . Bot. 1985, 63, 899-902. Norton, R.A.; Finlayson, A.J.; Towers, G.H.N. Phytochem. 1985, 24, 719-22. Kourany, E. M.S. Thesis, University of Ottawa, 1986, 170 pp. Elgersma, D.M.; Overeem, J.C. Neth. J . Plant Path. 1981, 87, 69-70. Thomas, C.A.; A l l e n , E.H. Phytopath. 1970, 60, 261-63. A l l e n , E.H.; Thomas, C.A. Phytochem. 1971, 10, 1579-82.

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A l l e n , E.H.; Thomas, C.A. Phytopath. 1971, 61, 1107-09. A l l e n , E.H.; Thomas, C.A. Phytopath. 1972, 62, 471-74. Fawcett, C.H.; Spencer, D.M.; Wain, R.L.; F a l l i s , A.G.; Jones, E.R.H.; Le Quan, M.; Page, C.B.; Thaller, V.; Shubrook, D.C.; Whitham, P.M. J . Chem. Soc. 1968, 2455-62. Hargreaves, J.A.; Mansfield, J.W.; Coxon, D.T; Price, K.R. Phytochem. 1976, 15, 1119-21. Hargreaves, J.A.; Mansfield, J.W.; Rossal, S. Physiol. Plant Path. 1977, 11, 227-42. Letcher, R.M.; Widdowson, D.A.; Deverall, B.J.; Mansfield, J.W. Phytochem. 1970, 9, 249-52. Downum, K.R.; Towers, G.H.N. J . Nat. Prod. 1983, 44, 98-103. Downum, K.R.; K e i l , D.J.; Rodriguez, E. Biochem. Syst. Ecol. 1985, 13, 109-13. DiCosmo, F.; Norton, R.; Towers, G.H.N. Naturwissenschaften 1982, 69S, 550-51. Garrod, B.; Lea, E.J.A. Lewis B.G Ne Phytologist 1979 83 463-71. Schulte, K.E.; Wulfhorst 285-98. Smith, D.A.; Banks, S.W. Phytochem. 1986, 25, 979-95. Bakker, J . ; Gommers, F.J.; Smits, L.; Fuchs, A.; de Vries, F.W. Photochem. Photobiol. 1983, 38, 323-29. Sutton, D.C.; G i l l a n , F.T.; Susie, M. Phytochem. 1985, 24, 2877-79. Kemp, M.S.; Burden, R.S.; L o e f f l e r , R.S.T. J . Chem. Soc. Perkin. Trans. I 1983, 2267-72. Kemp, M.S.; Burden, R.S. J . Chem. Soc. Perkin Trans. I 1984, 1441-43. Burden, R.S.; Kemp, M.S.; W i l t s h i r e , C.W. J . Chem. Soc. Perkin Trans. I 1984, 1445-48.

RECEIVED November 20, 1986

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 22

Photodynamic Herbicides and Chlorophyll Biosynthesis Modulators C. A. Rebeiz, A. Montazer-Zouhoor, J. M. Mayasich, B. C. Tripathy, S. M. Wu, and C. C. Rebeiz Laboratory of Plant Pigment Biochemistry and Photobiology, University of Illinois, Urbana, IL 61801

Higher plants hav ferent greening d i v i n y l protochlorophyllide biosynthetic patterns at night and i n daylight. We have succeeded i n demon s t r a t i n g that the photodynamic h e r b i c i d a l suscepti bility of a particular plant species depends on i t s greening group and on the chemical nature of the δ-aminolevulinic acid (ALA)-dependent tetrapyrroles that accumulate as a consequence of ALA-treatment. Three groups of chemicals which modulate differen tially the monovinyl and d i v i n y l monocarboxylie chlo rophyll biosynthetic routes have now been i d e n t i f i e d namely (a) enhancers of ALA conversion to monovinyl or d i v i n y l tetrapyrroles, (b) inducers of ALA forma tion and conversion to monovinyl and d i v i n y l t e t r a pyrroles and (c) i n h i b i t o r s of d i v i n y l tetrapyrrole conversion to monovinyl tetrapyrroles. By combining ALA with member(s) of one or more of the foregoing groups o f chlorophyll biosynthesis modulators, it has become possible to design h e r b i c i d a l formulations which are very s p e c i f i c to certain crop and weed plant species under a wide range of growth conditions.

I n 1984, a n o v e l approach f o r t h e d e s i g n o f u s e f u l h e r b i c i d e s was r e p o r t e d (V). The concept and phenomenology were i l l u s t r a t e d by the d e s c r i p t i o n o f an e x p e r i m e n t a l h e r b i c i d e based on a n a t u r a l l y o c c u r r i n g amino a c i d , 6 - a m i n o l e v u l i n i c a c i d ( A L A ) . S i n c e t h e n , c o n s i d e r a b l e p r o g r e s s has been a c h i e v e d i n expanding t h e scope o f t h i s e x p e r i m e n t a l h e r b i c i d a l system, i n u n d e r s t a n d i n g i t s mode o f a c t i o n and i n i t s development i n t o a v i a b l e h e r b i c i d e . Review o f t h e E x p e r i m e n t a l Photodynamic H e r b i c i d e System P r i n c i p l e s and G u i d e l i n e s . The d i s c o v e r y o f n o v e l p e s t i c i d e s has t r a d i t i o n a l l y been t h e r e s u l t o f b l i n d s c r e e n i n g , t h a t i s t h e 0097-6156/87/0339-0295$09.50/0 © 1987 American Chemical Society

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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r e s u l t o f e x p e r i m e n t a t i o n i n v o l v i n g t r i a l and e r r o r . I n a t y p i c a l year an a g r i c h e m i c a l company may b l i n d - s c r e e n 20,000 t o 50,000 c h e m i c a l s f o r h e r b i c i d a l o r i n s e c t i c i d a l a c t i v i t y . The v e r y few c h e m i c a l s t h a t e x h i b i t promise a r e then i n v e s t i g a t e d f u r t h e r and t h e i r e f f i c a c y , s e l e c t i v i t y , e n v i r o n m e n t a l impact and p h y t o t o x i c i t y a r e e v a l u a t e d . I n t h i s u n d e r t a k i n g , t h e u n d e r s t a n d i n g o f t h e mode o f a c t i o n o f a p a r t i c u l a r p e s t i c i d e i s u s u a l l y a s s i g n e d a low p r i o r i t y . Sometimes i t i s n e i t h e r i n v e s t i g a t e d n o r u n d e r s t o o d . In 1982 we s t a r t e d a r e s e a r c h e f f o r t aimed a t t h e d e s i g n o f n o v e l h e r b i c i d e s by a d o p t i n g an approach t o t a l l y d i f f e r e n t from t h e c o n v e n t i o n a l i n d u s t r i a l approach. The i d e a was t o draw on b a s i c b i o l o g i c a l knowledge i n o r d e r t o d e s i g n a h e r b i c i d e which was based on a p r e c o n c e i v e d mode o f a c t i o n . Development o f t h e Concept. The c o n c e p t u a l development o f t h e h e r b i c i d e was n a t u r a l l y i n f l u e n c e d by our past r e s e a r c h e x p e r i e n c e w i t h t h e c h e m i s t r y and b i o c h e m i s t r f th greenin Th g r e e n i n g p r o c e s s i s on b i o s p h e r e . The o t h e r f o u d u c t i o n , and growth d i f f e r e n t i a t i o n and development. I t i s most o b v i o u s i n t h e s p r i n g when deciduous a n n u a l and p e r e n n i a l p l a n t s a c q u i r e t h e i r green c o l o r . T h i s v i s u a l g r e e n i n g phenomenon i s a c h e m i c a l e x p r e s s i o n o f t h e b i o s y n t h e s i s and a c c u m u l a t i o n o f c h l o r o p h y l l ( C h i ) by d e v e l o p i n g c h l o r o p l a s t s . I t i s these green organ e l l e s which a r e r e s p o n s i b l e f o r t h e c o n v e r s i o n o f s o l a r energy t o c h e m i c a l energy v i a t h e p r o c e s s o f p h o t o s y n t h e s i s . Without t h e normal o c c u r r e n c e o f t h e g r e e n i n g p r o c e s s , p h o t o s y n t h e s i s i s n o t p o s s i b l e and o r g a n i c l i f e a s we know i t , i s n o t p o s s i b l e e i t h e r . S i n c e t h e g r e e n i n g phenomenon o c c u p i e s such a c e n t r a l p o s i t i o n i n t h e economy o f t h e b i o s p h e r e , we reasoned t h a t i t s h o u l d be q u i t e p o s s i b l e t o d e s i g n a h e r b i c i d e w i t h a mode o f a c t i o n r o o t e d i n t o some f a c e t s o f t h e g r e e n i n g p r o c e s s . T h i s i n t u r n r a i s e d t h e i m p o r t a n t q u e s t i o n o f which a s p e c t s o f t h e g r e e n i n g phenomenon would b e s t l e n d i t s e l f f o r such an u n d e r t a k i n g . We f i r s t c o n s i d e r e d t h e p o s s i b i l i t y o f d e s i g n i n g a h e r b i c i d e t h a t may i n t e r f e r e w i t h t h e b i o s y n t h e s i s o f C h i . Such a h e r b i c i d e would a c t by p r e v e n t i n g t h e t r e a t e d p l a n t s from r e p l e n i s h i n g t h e C h i o f t h e f u l l y developed l e a v e s and from f o r m i n g new C h i t o accommodate t h e expan s i o n o f new l e a v e s . We opted a g a i n s t t h i s s t r a t e g y because under f i e l d c o n d i t i o n s s e e d l i n g s emerge from t h e s o i l e s s e n t i a l l y green and t h e i r r a t e o f C h i b i o s y n t h e s i s i s as slow as t h e i r r a t e o f C h i t u r n o v e r . I n o t h e r words we c o n j e c t u r e d t h a t such a h e r b i c i d e would be a very slow a c t i n g h e r b i c i d e , p a r t i c u l a r l y on weeds t h a t had a l r e a d y a t t a i n e d a c e r t a i n s i z e . Another s t r a t e g y o f f e r e d more promise. We s p e c u l a t e d t h a t i f green p l a n t s c o u l d be induced t o accumulate massive amounts o f t e t r a p y r r o l e s , i . e . o f C h i p r e c u r s o r s , by s p r a y i n g them w i t h c e r t a i n c h e m i c a l s , t h e r e i s a good chance t h a t these compounds may be developed i n t o n o n - s e l e c t i v e h e r b i c i d e s . We opted f o r t h i s ap proach f o r s e v e r a l r e a s o n s . For one, t e t r a p y r r o l e s and i n p a r t i c u l a r M g - t e t r a p y r r o l e s , a r e n o t o r i o u s t y p e I I p h o t o s e n s i t i z e r s (1-3.)• They have t h e tendency t o absorb l i g h t energy and t o p h o t o s e n s i t i z e the f o r m a t i o n o f s i n g l e t oxygen. The l a t t e r i s a v e r y p o w e r f u l o x i d a n t and c a n t r i g g e r a f r e e r a d i c a l c h a i n r e a c t i o n t h a t c a n

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Photodynamic Herbicides

297

d e s t r o y b i o l o g i c a l membranes, n u c l e i c a c i d s , enzymes, and many o t h e r p r o t e i n s (3.)- F u r t h e r m o r e , m e t a b o l i c M g - t e t r a p y r r o l e s are e x t r e m e l y b i o d e g r a d a b l e (4.-7) and t h e i r e n v i r o n m e n t a l impact would, t h e r e f o r e , be n e g l i g i b l e . What was not known however, was whether a green p l a n t t h a t had a c q u i r e d i t s f u l l complement o f C h i and which was b i o s y n t h e s i z i n g C h i a t r a t e s commensurate w i t h i t s slow C h i t u r n - o v e r r a t e , c o u l d be i n d u c e d , by c h e m i c a l t r e a t m e n t , t o accumulate enough t e t r a p y r r o l e s t o cause photodynamic damage. The e l u c i d a t i o n o f t h i s i s s u e i n v o l v e d the d e t e r m i n a t i o n o f very s m a l l amounts o f t e t r a p y r r o l e s i n the presence o f very l a r g e amounts of C h i . F o r t u n a t e l y , t h i s d i f f i c u l t a n a l y t i c a l problem had been t a c k l e d and s o l v e d about 10 y e a r s e a r l i e r (8-11). This i n turn made i t p o s s i b l e t o t e s t and t o demonstrate the most i m p o r t a n t premise o f the proposed h e r b i c i d e concept, namely the p o s s i b l e i n d u c t i o n o f M g - t e t r a p y r r o l e a c c u m u l a t i o n and o f photodynamic damage i n green p l a n t s by c h e m i c a l t r e a t m e n t (1_). Choice of H e r b i c i d e . I c h o i c e of h e r b i c i d e became s t r a i g h t f o r w a r d . For y e a r s i t had been known t h a t dark-grown ( i . e . e t i o l a t e d ) p l a n t s accumulated s i g n i f i c a n t amounts o f t e t r a p y r r o l e s upon treatment w i t h ALA (12-14) ( F i g . 1). T h i s b e h a v i o u r had i t s o r i g i n i n t h r e e d i s t i n c t phenomena: (a) 6 - a m i n o l e v u l i n i c a c i d , a 5-carbon amino a c i d i s the p r e c u r s o r o f heme and C h i i n n a t u r e (1_5, 1_6^, (b) the f o r m a t i o n and a v a i l a b i l i t y o f ALA f o r heme and C h i f o r m a t i o n i s h i g h l y r e g u l a t e d by l i v i n g c e l l , (1_7, 1_8) and (c) s i n c e e t i o l a t e d p l a n t s c o n t a i n e d o n l y s m a l l amounts o f p r o t o c h l o r o p h y l l s ( P c h l s ) [ t h e immediate p r e c u r s o r s o f c h l o r o p h y l l i d e ( C h i w i t h o u t p h y t o l ) and o f C h i ] , but d i d not c o n t a i n any C h i (J_9)» the C h i b i o s y n t h e t i c pathway i n such p l a n t s was e x t r e m e l y p o t e n t ( 2 0 ) . I t was p o i s e d f o r f o r m i n g mas s i v e amounts o f C h i , s h o u l d t h e demand a r i s e upon e x p o s i n g the p l a n t s t o l i g h t (21_). Upon t r e a t m e n t o f such p l a n t s w i t h ALA, an i m p o r t a n t b i o s y n t h e t i c r e g u l a t o r y step was bypassed, namely the r e g u l a t i o n o f ALA f o r m a t i o n and a v a i l a b i l i t y t o the p l a n t ( 1 8 ) . Deluged w i t h l a r g e amounts o f ALA, the C h i b i o s y n t h e t i c machinery o f the e t i o l a t e d p l a n t s was f o r c e d t o c o n v e r t the ALA t o Mg-protop o r p h y r i n s and t o P c h l s i n d a r k n e s s and t o c o n v e r t some o f the l a t t e r t o c h l o r o p h y l l i d e s and t o C h i i n the l i g h t (20-22). As a consequence o f the above c o n s i d e r a t i o n s , and o f the known photody namic e f f e c t s o f t e t r a p y r r o l e s ( v i d e s u p r a ) , ALA appeared t o be the p e r f e c t c a n d i d a t e f o r a h e r b i c i d e . Furthermore, s i n c e ALA was a n a t u r a l amino a c i d t h a t o c c u r r e d i n a l l l i v i n g c e l l s and was an i n t e g r a l p a r t o f the food c h a i n , i t s e n v i r o n m e n t a l impact was expected t o be m i n i m a l . T h e r e f o r e , what remained t o be seen was whether mature green p l a n t s would r e a c t t o ALA t r e a t m e n t l i k e e t i o l a t e d p l a n t s and accumulate enough t e t r a p y r r o l e s t o undergo photodynamic damage. The d e m o n s t r a t i o n o f t h i s phenomenon was d e s c r i b e d i n (1_). D i s c o v e r y o f the S e l e c t i v e H e r b i c i d a l E f f e c t o f ALA. As was j u s t mentioned, the ALA - based h e r b i c i d e was meant t o be a n o n - s e l e c t i v e h e r b i c i d e . S i n c e i t a c t e d v i a the C h i b i o s y n t h e t i c pathway and s i n c e the l a t t e r was such a fundamental p r o c e s s which was be l i e v e d t o be common t o a l l green p l a n t s , we had no r e a s o n t o

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

298

LIGHT-ACTIVATED PESTICIDES

I PROTOCHLOROPHYLL a. R = _ C H = C H ; R 2

2

=_CH ;R

3

3

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4

f

b. R 2 = _ C H _ C H 3 ; R = _ C H ; R = F . A I i 2 _ M V 7 _ F A I . E 2

3

3

c. R 2 = - C H = C H ; R = _ C H ; R 2

3

3

d. R = _ C H = C H ; R = H ; R 2

2

3

4

4

4

2

3

2

2

3

3

3

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l

= H ; D V , 7 _ C O O H I O _ C 0 M e Pchlide l

2

l

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e. R = — C H 2 _ C H ; R = _ C H ; R f. R = _ C H _ C H ; R = H ; R

l

3

4

4

= H, 2 _ M V , 7 _ C O O H , I O _ C 0 M e , Pchlide 2

= A l k ; 2 _ M V , 7 _ A l k . E , I O - C O O H Pchlide

F i g u r e 1. S t r u c t u r e of m o n o v i n y l (MV) and d i v i n y l (DV) Mgp r o t o p o r p h y r i n monoester (MPE) and p r o t o c h l o r o p h y 1 1 i d e (Pchlide). (Reproduced w i t h p e r m i s s i o n from R e f e r e n c e 26. C o p y r i g h t 1983 N i j h o f f / D r . W. Junk P u b l i s h e r s . )

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

22.

I.

299

Photodynamic Herbicides

REBEIZ ET AL.

Mg PROTO diester, Mg PROTO monoester and M.g PROTO pools a. R = _ C H = C H ; R 3 = _ C H ; R = 2

2

3

b. R = _ C H _ C H i R = - . C H 2

2

3

3

F.AI;DV,7_FAI.E,6Me.P,Mg

4

P r o t o ( D V Mg Proto diester)

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3

4

c. R = - C H = C H ; R = _ C H i R = H ; D V , 7 _ C O O H 6 M e . P . M g

Proto(DV

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d. R = _ C H = C H ; R = H ; R = A l k ; D V 7 _ A l k . E , 6 _ C O O H M g

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2

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3

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l

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2

3

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2

3

3

2

2

h. R = _ C H _ C H ; R 2

2

2

3

l

MgProto 6 M E )

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3

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4

4

3

7ester)

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Figure 1.—Continued. S t r u c t u r e o f m o n o v i n y l (MV) and d i v i n y l (DV) M g - p r o t o p o r p h y r i n monoester (MPE) and p r o t o c h l o r o p h y 1 1 i d e (Pchlide). (Reproduced w i t h p e r m i s s i o n from R e f e r e n c e 26. C o p y r i g h t 1983 N i j h o f f / D r . W. Junk P u b l i s h e r s . )

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

300

LIGHT-ACTIVATED PESTICIDES

s u s p e c t even t h e p o s s i b i l i t y o f an ALA h e r b i c i d a l s e l e c t i v i t y . I t was, t h e r e f o r e , o u t o f s c i e n t i f i c r o u t i n e t h a t t h e h e r b i c i d a l e f f e c t o f ALA toward g r a s s y monocotyledonous p l a n t s (monocots) such as c o r n , wheat, o a t and b a r l e y was m o n i t o r e d . To our s u r p r i s e t h e t r e a t e d g r a s s y monocots were e s s e n t i a l l y u n a f f e c t e d by t h e s p r a y . T h i s prompted us t o expand t h e scope o f t h e A L A - s u s c e p t i b i l i t y s t u d i e s t p a v a r i e t y o f monocot and d i c o t y l e d o n o u s ( d i c o t ) p l a n t s . E s s e n t i a l l y , t h r e e t y p e s o f h e r b i c i d a l r e s p o n s e s , named Type I , I I and I I I , t o ALA + 2 , 2 - d i p y r i d y l (DPy), a C h i b i o s y n t h e s i s modula t o r , were noted (1_): ( a ) t y p e I r e s p o n s e was e x h i b i t e d by p l a n t s such as cucumber, which a f t e r t r e a t m e n t , d i e d v e r y r a p i d l y , (b) t y p e I I response was e x h i b i t e d by p l a n t s such as soybean which accumulated t e t r a p y r r o l e s i n t h e l e a f y t i s s u e s b u t n o t i n t h e stems and c o t y l e d o n s . Only t h e l e a v e s e x h i b i t e d photodynamic damage b u t the p l a n t s r e c o v e r e d and regrew v i g o r o u s l y , and ( c ) type I I I r e sponse was e x h i b i t e d by monocots such as c o r n , wheat, o a t and barley. Although the amounts o f t e t r a p y r r o l e s t r e a t e d s e e d l i n g s c o n t i n u e d t o grow and developed i n t o h e a l t h y plants. A l t h o u g h a t t h e t i m e , we d i d n o t u n d e r s t a n d t h e b i o c h e m i c a l o r i g i n o f t h i s d i f f e r e n t i a l response t o t h e ALA t r e a t m e n t , we i m m e d i a t e l y r e a l i z e d t h e importance o f t h i s phenomenon, and we undertook t h e t a s k o f e l u c i d a t i n g t h e m o l e c u l a r b a s i s o f t h i s unexpected p l a n t b e h a v i o u r . T h i s i n v o l v e d r e s e a r c h d e a l i n g w i t h the c h e m i c a l and b i o c h e m i c a l h e t e r o g e n e i t y o f t h e C h i b i o s y n t h e t i c pathway as w e l l as r e s e a r c h d e a l i n g w i t h d i f f e r e n c e s i n t h e greening patterns o f various higher plant species. The r e s u l t s o f t h i s r e s e a r c h e f f o r t a r e d e s c r i b e d below. f

The

Multibranched

C h i a. B i o s y n t h e t i c Pathway

On t h e b a s i s o f emerging e x p e r i m e n t a l e v i d e n c e , we had proposed i n 1 9 8 0 , t h a t t h e C h i b i o s y n t h e t i c pathway was n o t a s i n g l e , l i n e a r c h a i n o f r e a c t i o n s t h a t l e d t o t h e f o r m a t i o n o f one C h i a and one C h i b c h e m i c a l s p e c i e s as had been commonly b e l i e v e d ( 2 3 - 2 5 ) . I n s t e a d we s u g g e s t e d t h a t t h e e x p e r i m e n t a l e v i d e n c e was more com p a t i b l e with the operation of a multibranched Chi b i o s y n t h e t i c pathway which l e d t o t h e f o r m a t i o n o f s e v e r a l C h i a c h e m i c a l species, having d i f f e r e n t functions i n photosynthesis ( 2 5 ) . This h y p o t h e s i s was l a t e r on r e i n f o r c e d and expanded (1J3, 26). At t h a t time we had no r e a s o n t o s u s p e c t t h a t v a r i o u s p l a n t s p e c i e s may d i f f e r i n their Chi biosynthetic a c t i v i t i e s u n t i l the d i f f e r e n t i a l ALA h e r b i c i d a l r e s p o n s e was o b s e r v e d . The l a t t e r c o u l d be r e a d i l y e x p l a i n e d on t h e b a s i s o f d i f f e r e n c e s i n t h e C h i b i o s y n t h e t i c pathways among v a r i o u s p l a n t s p e c i e s . The i n v e s t i g a t i o n o f t h i s i s s u e was t h e r e f o r e c a r r i e d o u t w i t h i n t h e c o n c e p t u a l framework o f the m u l t i b r a n c h e d C h i a b i o s y n t h e t i c pathway ( 2 6 , 2 7 ) , and l e d t o the d i s c o v e r y o f t h e 4 g r e e n i n g p a t t e r n s o f p l a n t s which a r e de s c r i b e d below. The m u l t i b r a n c h e d pathway r e p r o d u c e d i n F i g . 2 , c o n s i s t s o f s i x p a r a l l e l b i o s y n t h e t i c r o u t e s numbered 1 t o 6 . Most o f t h e C h i i n n a t u r e i s a c t u a l l y formed v i a r o u t e s 2 and 5 which are t h e major m o n o v i n y l (MV) and d i v i n y l (DV) m o n o c a r b o x y l i c r o u t e s o f t h a t pathway. M o n o v i n y l m o n o c a r b o x y l i c t e t r a p y r r o l e s possess

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

22.

REBEIZ ET

AL.

Photodynamic Herbicides

one v i n y l and one f r e e c a r b o x y l i c group m o n o c a r b o x y l i c t e t r a p y r r o l e s possess two b o x y l i c group per m a c r o c y c l e ( F i g . 1 ) . t i o n s t h a t h e l p e d i n the f o r m u l a t i o n o f i n (28-39).

301 per m a c r o c y c l e w h i l e DV v i n y l and one f r e e c a r Some o f the key o b s e r v a t h a t pathway are d e s c r i b e d

C l a s s i f i c a t i o n o f Higher P l a n t s i n t o Four D i f f e r e n t Greening Groups As we j u s t mentioned ( v i d e s u p r a ) , t h e r e was no r e a s o n t o b e l i e v e t h a t green p l a n t s growing under n a t u r a l f i e l d c o n d i t i o n s , d i f f e r e d i n t h e i r C h i f o r m i n g pathways. Indeed, u n t i l v e r y r e c e n t l y we f i r m l y b e l i e v e d t h a t a l l green p l a n t s formed t h e i r C h i s i m u l t a n e o u s l y v i a the s i x C h i b i o s y n t h e t i c r o u t e s d e p i c t e d i n F i g . 2. This n o t i o n came under q u e s t i o n as a consequence o f two observations: (a) o f the f o r e m e n t i o n e d s p e c i e s - d e p e n d e n t d i f f e r e n t i a l ALA h e r b i c i d a l s u s c e p t i b i l i t y which c o u l d be e l e g a n t l y e x p l a i n e d by the occurrence of a d i f f e r e n t i a v a r i o u s p l a n t s p e c i e s an l a t e d ( i . e . dark-grown) p l a n t s p e c i e s d i d indeed d i f f e r i n t h e i r P c h l i d e and C h i b i o s y n t h e t i c c a p a b i l i t i e s d u r i n g t r e a t m e n t w i t h a l t e r n a t i n g l i g h t / d a r k pulses (36). B e f o r e i n v e s t i g a t i n g the d i f f e r e n t i a l o c c u r r e n c e o f v a r i o u s C h i b i o s y n t h e t i c r o u t e s among d i f f e r e n t p l a n t s p e c i e s , i t was mandatory, however, t o develop the n e c e s s a r y a n a l y t i c a l and p r e p a r a t o r y methodology. With the development o f the a p p r o p r i a t e e x p e r i m e n t a l method o l o g y (40, 4l_) i t became p o s s i b l e t o i n v e s t i g a t e the p u t a t i v e o c c u r rence o f a d i f f e r e n t i a l C h i b i o s y n t h e t i c h e t e r o g e n e i t y i n green p l a n t s . T h i s was a c h i e v e d by s i m p l y a n a l y z i n g the MV and DV t e t r a p y r r o l e c o n t e n t o f r o u t e s 1 and 6, r o u t e s 2 + 3 and r o u t e s 4+5 ( F i g . 2) i n v a r i o u s p l a n t s p e c i e s growing under n a t u r a l p h o t o p e r i o d i c growth c o n d i t i o n s . I t was c o n s i d e r e d t h a t the amount o f a s p e c i f i c MV or DV t e t r a p y r r o l e b e l o n g i n g t o a s p e c i f i c MV or DV b i o s y n t h e t i c r o u t e and which was d e t e c t a b l e a t any p a r t i c u l a r t i m e , was r e l a t e d t o the f l o w o f t e t r a p y r r o l e i n t e r m e d i a t e s v i a t h a t b i o s y n t h e t i c r o u t e a t t h a t p a r t i c u l a r t i m e . Because o f the c y c l i c a l t e r n a t i o n o f n i g h t ( d a r k n e s s ) and l i g h t ( d a y l i g h t ) i n n a t u r e , the MV and DV t e t r a p y r r o l e c o n t e n t o f the v a r i o u s p l a n t s p e c i e s was a n a l y z e d at two s t a g e s o f the p h o t o p e r i o d : (a) a t the end o f the dark phase o f the p h o t o p e r i o d and (b) i n the m i d d l e o f the l i g h t phase o f the p h o t o p e r i o d . The a n a l y s i s a t the end o f the dark phase o f the p h o t o p e r i o d was meant t o r e f l e c t the a c t i v i t y o f the b i o s y n t h e t i c r o u t e s at n i g h t , w h i l e a n a l y s i s i n the m i d d l e o f the day was meant t o r e f l e c t the a c t i v i t y o f the b i o s y n t h e t i c r o u t e s i n d a y l i g h t . I t was c o n j e c t u r e d t h a t s h o u l d d i f f e r e n c e s be observed among v a r i o u s p l a n t s p e c i e s w i t h r e s p e c t t o any two b i o s y n t h e t i c r o u t e s , as f o r example between the MV and DV r o u t e s i n d a r k n e s s (D) i . e . at n i g h t o r i n the l i g h t ( L ) , i . e . i n d a y l i g h t , f o u r meaning f u l b i o s y n t h e t i c c o m b i n a t i o n s may be o b s e r v e d , namely (a) dark d i v i n y l / l i g h t d i v i n y l (DDV/LDV), (b) DMV/LDV, ( c ) DDV/LMV and (d) DMV/LMV. I n the c o u r s e o f our i n v e s t i g a t i o n s a l l f o u r DV-MV P c h l i d e c o m b i n a t i o n s were observed ( v i d e i n f r a ) .

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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F i g u r e 2. S i x - b r a n c h e d Chla b i o s y n t h e t i c pathway: DV, d i v i n y l ; MV, m o n o v i n y l ; FA1, f a t t y a l c o h o l ; Phy, p h y t o l ; E, e s t e r ; ME, methyl e s t e r ; A l k , a l k y l group of unknown c h a i n l e n g t h ; Me, m e t h y l ; ALA, 6 - a m i n o l e v u l i n i c a c i d ; PBG, porpho b i l i n o g e n ; Urogen, u r o p o r p h y r i n o g e n ; Coprogen, c o p r o p o r p h y r i n o g e n ; Protogen, p r o t o p o r p h y r i n o g e n ; P r o t o , p r o t o p o r p h y r i n IX; LWMP, longer wavelength m e t a l l o p o r p h y r i n s ( t h e p u t a t i v e i n t e r m e d i a t e s o f r i n g E f o r m a t i o n ) ; P, e s t e r i f i c a t i o n w i t h g e r a n y l g e r a n i o l f o l l o w e d by stepwise c o n v e r s i o n o f the l a t t e r t o p h y t o l ; M, m e t h y l a t i o n . (Reproduced w i t h p e r m i s s i o n from Reference 26. C o p y r i g h t 1983 N i j h o f f / D r . W. Junk P u b l i s h e r s . )

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The DDV/LDV G r e e n i n g Group. P l a n t s p e c i e s such as cucumber (Cucumis s a t i v u s L . ) , common p u r s l a n e ( P o r t u l a c a o l e r a c e a ) and mustard ( B r a s s i c a Juncea L. and B r a s s i c a kaber) b e l o n g i n t h i s group ( 3 8 ) . D u r i n g t h e dark phase o f a 10 h dark/14 h l i g h t phot o p e r i o d , t h e s e p l a n t s accumulate m a i n l y DV p r o t o c h l o r o p h y l l i d e ( P c h l i d e ) and s m a l l e r amounts o f MV P c h l i d e (42, 4 3 ) . At daybreak, C h i f o r m a t i o n proceeds v i a t h e DV-enriched P c h l i d e p o o l ( 4 2 ) . L a t e r on d u r i n g t h e day, t h e p r o p o r t i o n o f MV P c h l i d e drops do a v e r y low l e v e l and C h i f o r m a t i o n proceeds m a i n l y v i a t h e DV-en r i c h e d Pchlide pool (42). The DMV/LDV G r e e n i n g Group. T h i s group appears t o be t h e l a r g e s t g r e e n i n g group o f h i g h e r p l a n t s and i n c l u d e monocots, such as c o r n (Zea mays L.) wheat ( T r l t i c u m s e c a l e L.) and b a r l e y (Hordeum v u l g a r e ) and d i c o t s such as t h e common bean ( P h a s e o l u s v u l g a r i s L . ) , soybean ( G l y c i n e max L.) and pigweed (Amaranthus r e t r o f l e x u s L.) ( 3 8 ) . At t h e b e g i n n i n t h e s e p l a n t s s h i f t ver p a t t e r n (which p r e v a i l daylight) biosyntheti p a t t e r n . During t h e n i g h t they accumulate m a i n l y MV P c h l i d e and very s m a l l amounts o f DV P c h l i d e (42, 43.). At daybreak, C h i forma t i o n proceeds v i a t h e MV e n r i c h e d P c h l i d e p o o l . Under n a t u r a l day l i g h t , t h e p l a n t s s h i f t back t o a DV P c h l i d e a c c u m u l a t i o n p a t t e r n and form C h i m a i n l y v i a t h e DV-enriched P c h l i d e p o o l (42, 4 3 ) . The DDV/LMV G r e e n i n g Group. T h i s r e c e n t l y d i s c o v e r e d g r e e n i n g group was f i r s t d e s c r i b e d i n 1986 (38) and (43) and so f a r i n c l u d e s fewer p l a n t s p e c i e s than t h e o t h e r t h r e e g r e e n i n g groups. I t s members i n c l u d e g i n k g o (Ginkgo b i l o b a ) and v i o l e t s p e c i e s ( V i o l a s p e c i e s ) . D u r i n g t h e dark phase o f t h e p h o t o p e r i o d , these p l a n t s accumulate m a i n l y DV P c h l i d e and s m a l l e r amounts o f MV P c h l i d e . At daybreak, they form C h i m a i n l y v i a t h e DV-enriched P c h l i d e p o o l and l a t e r on i n d a y l i g h t form C h i v i a t h e MV-enriched P c h l i d e p o o l . The DMV/LMV Greening Group. L i k e w i s e t h i s g r e e n i n g group was a l s o r e c e n t l y d e s c r i b e d (38, ^ 3 ) . I t i n c l u d e s p l a n t s p e c i e s such as a p p l e (Pyrus malus) and Johnson g r a s s (Sorghum h a l e p e n s e ) . During the dark phase o f t h e p h o t o p e r i o d t h e s e p l a n t s accumulate predomi n a n t l y MV P c h l i d e and s m a l l e r amounts o f DV P c h l i d e . At daybreak and l a t e r on d u r i n g d a y l i g h t they form C h i m a i n l y v i a t h e MVe n r i c h e d P c h l i d e p o o l (38, 4 3 ) . Molecular

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S i n c e we s t r o n g l y s u s p e c t e d t h a t t h e d i f f e r e n t i a l ALA-dependent photodynamic s u s c e p t i b i l i t y o f green p l a n t s was c l o s e l y t i e d t o t h e biochemical o r i g i n o f the d i f f e r e n t i a l greening patterns o f higher p l a n t s , t h i s r e l a t i o n s h i p was n e x t i n v e s t i g a t e d . B i o s y n t h e t i c O r i g i n o f t h e DV and MV P c h l i d e A c c u m u l a t i o n P a t t e r n s i n t h e DDV/LDV Greening Group o f P l a n t s . The o r i g i n o f t h e DV P c h l i d e a c c u m u l a t i o n p a t t e r n i n t h i s g r e e n i n g group was r e a d i l y demonstrated w i t h t h e use o f t h e DDV/LDV cucumber c e l l - f r e e system

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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d e s c r i b e d i n (4J_). I t was shown t o o r i g i n a t e i n b i o s y n t h e t i c routes 2 + 3 ( F i g . 2). T h i s was a c h i e v e d by d e m o n s t r a t i n g t h e c o n v e r s i o n o f ALA t o DV P c h l i d e v i a DV p r o t o p o r p h y r i n ( P r o t o ) , DV Mg-Proto and DV Mg-Proto monoester i n d a r k n e s s (38., 39). A more i m p o r t a n t i s s u e , however, was whether t h e MV P c h l i d e a c c u m u l a t i o n p a t t e r n o r i g i n a t e d i n t h e c o n v e r s i o n o f DV P c h l i d e t o MV P c h l i d e o r whether I t o r i g i n a t e d somewhere e l s e . T h i s q u e s t i o n was s e t t l e d by d e m o n s t r a t i n g t h e b i o s y n t h e s i s o f MV P c h l i d e v i a t h e MV m o n o c a r b o x y l i c r o u t e s 4 + 5 ( F i g . 2). I t was shown t h a t a l though t h e cucumber e t i o c h l o r o p l a s t s system was n o t c a p a b l e o f c o n v e r t i n g DV P c h l i d e t o MV P c h l i d e , i t d i d c o n v e r t , v e r y e f f i c i e n t l y ALA t o MV P c h l i d e v i a MV P r o t o , MV Mg-Proto and MV Mg-Proto monoester (38, 39). F u r t h e r m o r e , i t was shown t h a t i n t h i s system, DV r o u t e s 2 + 3 and MV r o u t e s 4 + 5 ( F i g . 2) were ( a ) e i t h e r n o t i n t e r c o n n e c t e d , i . e . a t t h e l e v e l o f DV P c h l i d e , o r (b) were very weakly i n t e r c o n n e c t e d a t s i t e ( s ) between DV P r o t o and DV P c h l i d e (38, 39). The r e g u l a t i o n o f g r e e n i n g group o f p l a n t s i s p r e s e n t l y under i n v e s t i g a t i o n . B i o s y n t h e t i c O r i g i n o f t h e DV and MV P c h l i d e A c c u m u l a t i o n P a t t e r n s i n t h e DMV/LDV Greening Group. The o r i g i n o f t h e DV P c h l i d e accumu l a t i o n p a t t e r n i n t h i s g r e e n i n g group was i n v e s t i g a t e d w i t h t h e DMV/LDV b a r l e y c e l l - f r e e system d e s c r i b e d i n (41). The o r i g i n o f the DV P c h l i d e a c c u m u l a t i o n p a t t e r n was shown t o r e s i d e i n b i o s y n t h e t i c routes 2 + 3 ( F i g . 2) by d e m o n s t r a t i n g t h e c o n v e r s i o n o f ALA t o DV P c h l i d e v i a DV P r o t o , DV Mg-Proto and DV Mg-Proto monoester (38, 39). The o r i g i n o f t h e MV P c h l i d e a c c u m u l a t i o n p a t t e r n was however c o n s i d e r a b l y more complex than i n DDV/LDV p l a n t s . About 30% o f t h e MV P c h l i d e appeared t o be formed from ALA v i a t h e MV m o n o c a r b o x y l i c routes, i . e . routes 4 + 5 ( F i g . 2). T h i s was e v i d e n c e d by t h e d a r k - c o n v e r s i o n o f ALA t o MV P c h l i d e v i a MV P r o t o , MV Mg-Proto and MV Mg-Proto monoester i n b a r l e y e t i o c h l o r o p l a s t s p o i s e d i n t h e MV P c h l i d e a c c u m u l a t i o n mode (38, 39). A s i z a b l e f r a c t i o n o f t h e MV P c h l i d e p o o l appeared t o be a l s o formed from DV P r o t o , DV Mg P r o t o and DV Mg P r o t o monoester b u t n o t from DV P c h l i d e (38, 39). This was a p p a r e n t l y a c c o m p l i s h e d v i a one o r more DV t e t r a p y r r o l e r e d u c t a s e (s) t h a t c o n v e r t e d DV t e t r a p y r r o l e s t o MV t e t r a p y r r o l e s by r e d u c t i o n o f t h e v i n y l group a t p o s i t i o n 4 o f t h e m a c r o c y c l e t o an e t h y l group ( F i g . 1). As a consequence, i n t h i s g r e e n i n g group o f p l a n t s , t h e DV and MV m o n o c a r b o x y l i c b i o s y n t h e t i c r o u t e s were very s t r o n g l y i n t e r c o n n e c t e d (38, 39). The p r e c i s e number and b i o c h e m i c a l s i t e ( s ) o f t h e DV t e t r a p y r r o l e r e d u c t a s e s i s p r e s e n t l y under i n v e s t i g a t i o n . Very r e c e n t d a t a a l s o i n d i c a t e s t h a t i n DMV/LDV p l a n t s , under c e r t a i n g r e e n i n g c o n d i t i o n s , a s m a l l f r a c t i o n o f t h e DV P c h l i d e p o o l may be c o n v e r t i b l e t o MV P c h l i d e v i a a DV P c h l i d e r e d u c t a s e (B. C. T r i p a t h y and C. A. R e b e i z , u n p u b l i s h e d ) . I n v e s t i g a t i o n o f t h e r e g u l a t i o n o f t h e MV and DV monocarboxy l i c r o u t e s i n DMV/LDV p l a n t s i s i n p r o g r e s s . L i k e w i s e , t h e b i o s y n t h e t i c o r i g i n o f t h e DV and MV P c h l i d e a c c u m u l a t i o n p a t t e r n s i n t h e o t h e r two g r e e n i n g groups o f p l a n t s , i . e . i n t h e DDV/LMV and t h e DMV/LMV group i s a l s o under i n v e s t i g a t i o n .

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B i o c h e m i c a l O r i g i n o f t h e D i f f e r e n t i a l ALA-dependent Photodynamic S u s c e p t i b i l i t y o f Green P l a n t s The m o l e c u l a r b a s i s o f t h e d i f f e r e n t i a l photodynamic s u s c e p t i b i l i t y o f v a r i o u s p l a n t t i s s u e s and p l a n t s p e c i e s t o ALA t r e a t m e n t was i n v e s t i g a t e d w i t h i n t h e framework o f t h e f o l l o w i n g h y p o t h e s i s : (a) t h a t t h e a c c u m u l a t i o n o f t e t r a p y r r o l e s by A L A - t r e a t e d t i s s u e s was a necessary but not a s u f f i c i e n t c o n d i t i o n f o r the occurrence o f photodynamic damage and (b) t h a t i n t h e event o f t e t r a p y r r o l e a c c u m u l a t i o n t h e o c c u r r e n c e and e x t e n t o f photodynamic damage was l i k e l y t o depend (a) on t h e e x t e n t o f t e t r a p y r r o l e a c c u m u l a t i o n , (B) on t h e g r e e n i n g group o f t h e t r e a t e d p l a n t and (Y) on t h e chemi c a l n a t u r e o f t h e accumulated t e t r a p y r r o l e . I t was a l s o r e c o g n i z e d t h a t t h e e x t e n t o f a p l a n t s p e c i e s photodynamic s u s c e p t i b i l i t y t o ALA t r e a t m e n t may be due t o one o r more o f t h e f o r e m e n t i o n e d c o n d i t i o n s . The l o g i s t i c s behind t h e above h y p o t h e s i s was based upon the f o l l o w i n g o b s e r v a t i o n s The proposed n e c e s s i t o c c u r r e n c e o f photodynamic damage i s a consequence o f t h e b a s i c mode o f a c t i o n o f ALA toward s u s c e p t i b l e p l a n t s p e c i e s . Indeed t h e r e l a t i o n s h i p between ALA t r e a t m e n t , t o t a l t e t r a p y r r o l e a c c u m u l a t i o n and photodynamic damage has a l r e a d y been demonstrated w i t h s u s c e p t i b l e p l a n t s p e c i e s such as cucumber (1_). On t h e o t h e r hand, t h e p r o p o s a l o f t h e " n o n - s u f f i c i e n c y " c o n d i t i o n was on t h e b a s i s t h a t a l t h o u g h some t r e a t e d p l a n t s p e c i e s accumulated l a r g e amounts o f t e t r a p y r r o l e s , they d i d not undergo s i g n i f i c a n t photodynamic damage (I). I n s u s c e p t i b l e p l a n t s t h a t responded t o ALA t r e a t m e n t by a c c u m u l a t i n g t e t r a p y r r o l e s , t h e proposed dependence o f photodynamic damage on t h e e x t e n t o f t e t r a p y r r o l e a c c u m u l a t i o n i s a g a i n an o b v i o u s consequence o f t h e demonstrated dependence o f photodynamic damage on t o t a l t e t r a p y r r o l e a c c u m u l a t i o n (1_). F i n a l l y i n p l a n t s p e c i e s c a p a b l e o f ALA-dependent t e t r a p y r r o l e a c c u m u l a t i o n , t h e proposed dependence o f photodynamic damage upon the g r e e n i n g group o f t h e t r e a t e d p l a n t as w e l l as upon t h e chemi c a l n a t u r e o f t h e accumulated t e t r a p y r r o l e was based on e x p e r i m e n t a l e v i d e n c e t h a t w i l l be d e s c r i b e d below. Dependence o f Photodynamic Damage on t h e E x t e n t o f T e t r a p y r r o l e A c c u m u l a t i o n : Case Study o f t h e D i f f e r e n t i a l Photodynamic S u s c e p t i b i l i t y o f Soybean C o t y l e d o n s and P r i m a r y Leaves t o ALA Treatment. T h i s case s t u d y e x p l o r e s t h e m o l e c u l a r b a s i s o f t h e d i f f e r e n t i a l photodynamic s u s c e p t i b i l i t y o f soybean c o t y l e d o n s and soybean p r i m a r y l e a v e s t o ALA-treatment. As may be r e c a l l e d , a l t h o u g h t h e p r i m a r y l e a v e s o f soybean s e e d l i n g s a r e v e r y s u s c e p t i b l e t o ALA t r e a t m e n t , soybean stems and c o t y l e d o n s a r e not (1_). As a conse quence, a l t h o u g h t h e p r i m a r y l e a v e s o f A L A - t r e a t e d s e e d l i n g s d i e w i t h i n a few hours o f exposure t o d a y l i g h t , t h e i n t a c t stems and c o t y l e d o n s s u s t a i n t h e p r o d u c t i o n o f new l e a v e s and t h e t r e a t e d s e e d l i n g s soon r e c o v e r . The r e s i s t a n c e o f soybean stems t o ALA t r e a t m e n t i s o b v i o u s l y r e l a t e d t o t h e l a c k o f t e t r a p y r r o l e a c c u m u l a t i o n by t h e t r e a t e d stems as d e s c r i b e d i n (1_). I n o r d e r t o determine whether t h e response o f soybean c o t y l e d o n s , a DMV/LDV t i s s u e ( 4 2 ) , t o ALA

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Herbicides

307

t r e a t m e n t was a l s o r o o t e d i n a l a c k o f t e t r a p y r r o l e a c c u m u l a t i o n , the f o l l o w i n g experiment was performed. Greenhouse grown soybean s e e d l i n g s were t r e a t e d w i t h a 5 mM ALA + 15 mM DPy s o l u t i o n p r e c i s e l y as d e s c r i b e d i n (1_). A f t e r wrapping t h e p l a n t s i n aluminum f o i l and dark i n c u b a t i o n f o r 17 h (1_), t e t r a p y r r o l e a c c u m u l a t i o n by the p r i m a r y l e a v e s and by t h e c o t y l e d o n s was determined and the s e e d l i n g s were exposed t o d a y l i g h t i n t h e greenhouse t o induce photodynamic damage. I n t h i s experiment t o t a l ALA-dependent t e t r a p y r r o l e a c c u m u l a t i o n by t h e p r i m a r y l e a v e s amounted t o 201 nmoles per 100 mg o f t i s s u e p r o t e i n w h i l e t h e c o t y l e d o n s accumulated o n l y 11 nmoles o f t e t r a p y r r o l e s per 100 mg p r o t e i n . A f t e r a few hours i n d a y l i g h t , photodynamic damage t o t h e l e a v e s amounted t o 100$ w h i l e t h e c o t y l e d o n s were u n a f f e c t e d . A l t o g e t h e r , t h e s e r e s u l t s i n d i c a t e d t h a t t h e l a c k o f photo dynamic damage t o soybean c o t y l e d o n s was due t o poor exogenous ALA-dependent t e t r a p y r r o l e a c c u m u l a t i o n by t h i s t i s s u e . Dependence o f Photodynami Accumulated T e t r a p y r r o l e and on t h e Greening Group o f t h e Treated P l a n t s . I n t h e s e p r e l i m i n a r y s t u d i e s , o n l y t h r e e model p l a n t systems have been used: ( a ) cucumber s e e d l i n g s , i n t h e c o t y l e d o n s t a g e as a r e p r e s e n t a t i v e o f t h e DDV/LDV group o f p l a n t s and (b) c o r n , and t o a l e s s e r e x t e n t soybean s e e d l i n g s , as monocot and d i c o t r e p r e s e n t a t i v e s o f t h e DMV/LDV g r e e n i n g group. The t e n t a t i v e c o n c l u s i o n s drawn from t h e s e s t u d i e s a r e , t h e r e f o r e , l i m i t e d i n scope and i n t h e f u t u r e may have t o be a d j u s t e d t o accpmmodate a d d i t i o n a l o b s e r v a t i o n s d e r i v e d from A L A - s u s c e p t i b i l i t y s t u d i e s w i t h DDV/LMV, DMV/LMV as w e l l as from a d d i t i o n a l DDV/LDV and DMV/LDV p l a n t s p e c i e s . In order t o c o r r e l a t e the accumulation o f s p e c i f i c t e t r a p y r r o l e s w i t h i n d u c t i o n o f photodynamic damage, we have used a group o f 13 c h e m i c a l s which a c t I n c o n c e r t w i t h ALA. The mode o f a c t i o n o f t h e s e c h e m i c a l s , which w i l l be r e f e r r e d t o as "modula t o r s " o f C h i b i o s y n t h e s i s , w i l l be d i s c u s s e d i n some d e t a i l s l a t e r on ( v i d e i n f r a ) . They were a c o n v e n i e n t t o o l i n d e m o n s t r a t i n g r e l a t i o n s h i p s between t h e a c c u m u l a t i o n o f s p e c i f i c t e t r a p y r r o l e s and photodynamic damage. Indeed, when used i n c o n c e r t w i t h ALA, they r e s u l t e d i n t h e preponderant a c c u m u l a t i o n o f s p e c i f i c MV o r DV t e t r a p y r r o l e s as d e s c r i b e d below. I n t h e s e e x p e r i m e n t s we used low c o n c e n t r a t i o n s o f ALA (5 mM) i n c o n j u n c t i o n w i t h h i g h e r c o n c e n t r a t i o n s (10 t o 30 mM) o f each one o f t h e 13 C h i b i o s y n t h e s i s m o d u l a t o r s . The i d e a was t o induce o n l y l i m i t e d photodynamic damage i n order t o c o r r e l a t e more p r e c i s e l y between t h e e x t e n t o f t h e l a t t e r and t h e a c c u m u l a t i o n o f s p e c i f i c t e t r a p y r r o l e s . The r e s u l t s o f t h e s e e x p e r i m e n t s a r e summarized below. Case Study 1: I n d u c t i o n o f Photodynamic Damage by ALA-dependent A c c u m u l a t i o n o f MV P c h l i d e i n Cucumber, a DDV/LDV P l a n t S p e c i e s b u t not i n Corn a DDMV/LDV P l a n t S p e c i e s . I n seven o f t h e t h i r t e e n d i f f e r e n t t r e a t m e n t s which used ALA i n c o n j u n c t i o n w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f i n d i v i d u a l members o f t h e 13 C h i b i o s y n t h e s i s m o d u l a t o r s , MV P c h l i d e was t h e preponderant t e t r a p y r r o l e t h a t accumulated i n t h e dark i n t h e t r e a t e d cucumber s e e d l i n g s . I n

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

308

LIGHT-ACTIVATED PESTICIDES

e v e r y one o f t h o s e 7 t r e a t m e n t s t h e b e s t c o r r e l a t i o n was observed between MV P c h l i d e d a r k - a c c u m u l a t i o n and photodynamic damage. One such experiment i s d e s c r i b e d i n Table IA. These r e s u l t s i n d i c a t e d t h a t cucumber a DDV/LDV p l a n t s p e c i e s was p h o t o d y n a m i c a l l y s u s c e p t i b l e t o ALA-dependent dark a c c u m u l a t i o n o f MV P c h l i d e . I n o r d e r t o determine whether t h i s c o n c l u s i o n was a l s o v a l i d f o r DMV/LDV p l a n t s p e c i e s , s i m i l a r experiments were performed on c o r n . I n a l l t h e s e e x p e r i m e n t s , t h e ALA-dependent dark-accumula t i o n o f MV P c h l i d e r e s u l t e d e i t h e r i n t h e absence o f photodynamic damage, as d e s c r i b e d i n Table I B o r i n very minor damage from which the s e e d l i n g s r e c o v e r e d v e r y r a p i d l y a s r e p o r t e d i n (1_). As a consequence o f t h e s e r e s u l t s we propose t h a t w h i l e DDV/LDV p l a n t s p e c i e s such as cucumber a r e p h o t o d y n a m i c a l l y s u s c e p t i b l e t o ALA-dependent MV P c h l i d e dark a c c u m u l a t i o n , DMV/LDV p l a n t s p e c i e s such as c o r n a r e e i t h e r n o t s u s c e p t i b l e o r much l e s s p h o t o d y n a m i c a l l y s u s c e p t i b l e than t h e DDV/LDV p l a n t s p e c i e s . T h i s hypothesis i s presentl species belonging to th Case Study 2: I n d u c t i o n o f ALA-dependent DV P c h l i d e Dark Accumula t i o n Cause Less Photodynamic Damage i n Cucumber a DDV/LDV P l a n t S p e c i e s than i n Soybean, a DMV/LDV P l a n t S p e c i e s . Four o f t h e t h i r t e e n C h i b i o s y n t h e s i s modulators r e s u l t e d i n t h e dark-accumula t i o n o f more DV P c h l i d e than MV P c h l i d e i n p a r t i c u l a r a t t h e h i g h e r c o n c e n t r a t i o n range (20 and 30 mM) o f t h e m o d u l a t o r s . I n these e x p e r i m e n t s , a l t h o u g h t h e i n c i d e n c e o f photodynamic damage d i d c o r r e l a t e w i t h ALA-induced DV P c h l i d e dark a c c u m u l a t i o n ( T a b l e I C ) , the e x t e n t o f photodynamic damage was u s u a l l y l e s s pronounced than when cucumber was f o r c e d t o accumulate MV P c h l i d e ( T a b l e I I ) . I t i s n o t known a t t h i s s t a g e whether t h e reduced photodynamic damage induced by modulators t h a t cause t h e preponderant d a r k - a c c u m u l a t i o n o f DV P c h l i d e i n cucumber i s due t o a lower photodynamic s u s c e p t i b i l i t y o f DDV/LDV p l a n t s p e c i e s per u n i t o f accumulated DV P c h l i d e or t o some o t h e r c a u s e s . I n o r d e r t o determine whether DMV/LDV p l a n t s p e c i e s e x h i b i t a h i g h e r photodynamic s u s c e p t i b i l i t y t o DV P c h l i d e d a r k - a c c u m u l a t i o n than DDV/LDV p l a n t s p e c i e s , s i m i l a r experiments a r e now b e i n g performed on soybean s e e d l i n g s i n t h e p r i m a r y l e a f s t a g e . P r e l i m i n a r y r e s u l t s have so f a r i n d i c a t e d t h a t primary l e a v e s o f soybean a r e e x t r e m e l y s u s c e p t i b l e t o ALA-based t r e a t m e n t s t h a t do r e s u l t i n DV P c h l i d e a c c u m u l a t i o n i n cucumber. At t h i s s t a g e we have no r e a s o n t o doubt t h e c o r r e l a t i o n o f t h i s photodynamic s u s c e p t i b i l i t y w i t h DV P c h l i d e a c c u m u l a t i o n by t h e soybean p r i m a r y leaves. Case Study 3: I n d u c t i o n o f Photodynamic Damage by ALA-Dependent DV M g - P r o t o p o r p h y r i n (Monoester) Accumulation i n Both Cucumber and Corn. Two o f t h e 13 C h i b i o s y n t h e s i s modulators caused t h e massive ALA-dependent a c c u m u l a t i o n o f DV M g - p r o t o p o r p h y r i n (monoester) [MP(E)] i n cucumber, a DDV/LDV p l a n t s p e c i e s , and i n c o r n , a DMV/LDV p l a n t s p e c i e s . I n b o t h s p e c i e s i t i s t h e a c c u m u l a t i o n o f DV MP(E) t h a t e x h i b i t e d t h e b e s t c o r r e l a t i o n w i t h photodynamic damage ( T a b l e I , D, E ) . Corn, however, r e c o v e r e d a f t e r a few days

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Experiment

Cucumber

Plant Species

0

Solvent only 5 mM ALA 10 mM 2 - p y r i d i n e a l d o x i m e 5 mM ALA + 10 mM 2 - p y r i d i n e a l d o x i m e 20 mM 2 - p y r i d i n e aldoxime 5 mM ALA + 20 mM 2 - p y r i d i n e a l d o x i m e 30 mM 2 - p y r i d i n e a l d o x i m e 5 mM ALA + 30 mM 2 - p y r i d i n e a l d o x i m e Correlation coefficient Level of s i g n i f i c a n c e

Treatment

0 43 0 83 0 83 0 68

(S)

Photodynamic Damage

DV

MV

3

DV

MP(E)

0.00 51.38 4.55 44.64 12.16 35.69 17.60 65.08 0.817 5%

0.00 0.38 0.13 0.77 -0.50b 0.44 0.16 0.32

0.00 -0.60 -0.84 -0.52 -0.34 0.08 0.43 -0.20

Continued on next page

0.00 17.81 3.08 4.96 1.94 8.12 3.34 17.62 0.569 n.s.

Exogenous ALA-dependent tetrapyrrole accumulation i n nmoles p e r 100 mg p r o t e i n s

MV

Pchlide

S e e d l i n g s were sprayed i n t h e l a t e a f t e r n o o n w i t h s o l v e n t o n l y o r w i t h s o l v e n t c o n t a i n i n g 5 mM ALA (130 g/acre) and a modulator (10 t o 30 mM) a t a r a t e o f 40 g a l l o n s p e r a c r e : The s o l v e n t c o n s i s t e d o f a c e t o n e : e t h y l a c e t a t e : tween 80: H2O (45:45:1:90 v / v / v / v ) . The sprayed p l a n t s were wrapped i n aluminum f o i l and p l a c e d i n d a r k n e s s a t 28°C f o r about 17 h. The next morning, t h e t r e a t e d p l a n t s were sampled f o r t e t r a p y r r o l e a n a l y s i s t h e n were exposed t o d a y l i g h t i n t h e greenhouse f o r t h e e v a l u a t i o n o f photodynamic damage. For more d e t a i l s c o n s u l t ( 1 ) . P c h l i d e = p r o t o c h l o r o p h y l l i d e ; MP(E) - Mg p r o t o + Mg p r o t o monoester; MV = m o n o v i n y l ; DV = d i v i n y l ; n.s. = n o t s i g n i f i c a n t . Adapted from ( 2 7 ) .

Table I . D i f f e r e n c e s i n ALA-Dependent T e t r a p y r r o l e A c c u m u l a t i o n and Photodynamic Damage Between DDV/LDV and DMV/LDV P l a n t S p e c i e s

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Experiment

Corn

Plant Species

Solvent only 5 mM ALA 10 mM p i c o l i n i c a c i d 5 mM ALA + 10 mM p i c o l i n i c 20 mM p i c o l i n i c a c i d 5 mM ALA + 20 mM p i c o l i n i c 30 mM p i c o l i n i c a c i d 5 mM ALA + 30 MM p i c o l i n i c Correlation coefficient Level o f s i g n i f i c a n c e

Treatment

acid

acid

acid

0 0 0 0 0 0 0 0

(%)

DV

MV

0.00 17.36 39.86 16.03 49.93 10.24 58.49 81.29 0.000 n.s.

0.00 -2.30 -2.73 -1.83 15.79 -1.69 0;20 4.10 0.000 n.s.

0.00 -5.07 -0.06 -1.13 8.14 5.07 2.52 0.04 0.000 n.s.

0.00 -1.13 0.85 1.64 8.12 17.01 20.24 20.67 0.000 n.s.

3

DV

MP(E)

Exogenous ALA-dependent tetrapyrrole accumulation i n nmoles per 100 mg p r o t e i n s

MV

Pchlide

A c c u m u l a t i o n and Photodynamic

Photodynamic Damage

Table I . C o n t i n u e d . D i f f e r e n c e s i n ALA-Dependent T e t r a p y r r o l e Damage Between DDV/LDV and DMV/LDV P l a n t S p e c i e s

n S m

H

c/3

m

no

O

1 m

r 0 3C •7*

o

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Cucumber

Cucumber

l

1

d

0.00 -0.27 -0.41 8.52

0.00 12.20 40.45 98.32 0 0 0 30

0.00 -2.60 -1.85 39.04

0.00 0.18 48.47 75.20 56.77 85.38 45.92 44.43 0.861 n

0.00 0.18 -0.58 -0.22 1.48 0.41 0.13 0.92

C o n t i n u e d on next pag

0.00 9.52 1.53 20.01

0.00 -0.04 4.44 -0.11 -0.11 1.12 0.95 6.03 0.364 n.s.

0.00 3.15 9.09 23.38 8.78 32.69 7.92 14.12 0.623 20% 0.00 15.48 25.25 34.35 12.93 33.16 10.20 8.08 0.413 n.s.

0 10 73 90 93 93 100 100

Solvent only 5 mM ALA 10 mM 1 , 1 0 - p h e n a n t h r o l i n e 5 mM ALA + 10 mM 1 , 1 0 - p h e n a n t h r o l i n e 20 mM 1 , 1 0 - p h e n a n t h r o l i n e 5 mM ALA + 20 mM 1 , 1 0 - p h e n a n t h r o l i n e 30 mM 1 , 1 0 - p h e n a n t h r o l i n e 5 mM ALA + 30 mM 1 , 1 0 - p h e n a n t h r o l i n e Correlation coefficient Level of s i g n i f i c a n c e

Solvent only 5 mM ALA 10 mM 2 , 2 - d i p y r i d y l 5 mM ALA + 10 mM 2 , 2 - d i p y r i d y l

0.00 0.33 1.44 0.45 -0.34 -0.16 0.36 1.88

0.00 6.41 6.94 8.45 4.26 9.37 8.72 15.30 0.753 5%

0.00 30.62 10.50 11.07 1.49 7.32 2.52 1.73 0.29 n.s.

0 55 5 45 33 50 43 63

Solvent only 5 mM ALA 10 mM 1 , 7 - p h e n a n t h r o l i n e 5 mM ALA + 10 mM 1 , 7 - p h e n a n t h r o l i n e 20 mM 1 , 7 - p h e n a n t h r o l i n e 5 mM ALA + 20 mM 1 , 7 - p h e n a n t h r o l i n e 30 mM 1 , 7 - p h e n a n t h r o l i n e 5 mM ALA + 30 mM 1 , 7 - p h e n a n t h r o l i n e Correlation coefficient Level of s i g n i f i c a n c e

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Corn

Plant Species

,

20 mM 2 , 2 - d i p y r i d y l 5 mM ALA + 2 , 2 • - d i p y r i d y l 30 mM 2 , 2 ' - d i p y r i d y l 5 mM ALA + 30 mM 2 , 2 • - d i p y r i d y l Correlation coefficient Level of s i g n i f i c a n c e

Treatment

0 31 40 80

{%)

Photodynamic Damage DV

MV

3

DV

11.01 34.70 104.28 12.87 0.377 n.s.

1.01 -0.65 -1.18 -0.39 0.068 n.s.

4. 79 5. 12 7. 26 13. 54 0. 557 n .s.

1.60 7.33 9.41 25.13 0.700 10$

Exogenous ALA-dependent tetrapyrrole accumulation i n nmoles per 100 mg p r o t e i n s

MV

MP(E)

d

c

I s t h e d i f f e r e n c e between t h e t e t r a p y r r o l e c o n t e n t o f t h e ALA o r ALA + m o d u l a t o r - t r e a t e d p l a n t s and t h a t o f the c o n t r o l p l a n t s w h i c h were sprayed w i t h s o l v e n t o n l y . ^ N e g a t i v e v a l u e s i n d i c a t e a drop i n c o n t e n t i n comparison t o t h e c o n t e n t o f t h e c o n t r o l p l a n t s . R e f e r s t o t h e p r o b a b i l i t y t h a t f o r a p o p u l a t i o n f o r which t h e c o r r e l a t i o n c o e f f i c i e n t ( r ) i s e q u a l t o z e r o , a sample o f s i z e n can be t a k e n , f o r w h i c h t h e c o r r e l a t i o n e q u a l s o r exceeds t h e c a l c u l a t e d v a l u e o f r which i s r e p o r t e d i n t h e t a b l e f o r a g i v e n sample. S i n c e c o r n p l a n t s r e c o v e r e d from photodynamic damage, t h e v a l u e s r e p o r t e d f o r c o r n were d e t e r m i n e d two days after spraying. Those r e p o r t e d f o r cucumber were d e t e r m i n e d 10 days a f t e r s p r a y i n g .

a

Experiment

Pchlide

Table I . C o n t i n u e d . D i f f e r e n c e s i n ALA-Dependent T e t r a p y r r o l e A c c u m u l a t i o n and Photodynamic Damage Between DDV/LDV and DMV/LDV P l a n t S p e c i e s

C/3

5 m

n

H

C/J

m

O

1

H

0

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

2-pyridine aldehyde

Chlorophyll biosynthesis modulator

Solvent only 5 mM ALA 10 mM modulator 5 mM ALA + 10 mM modulator 20 mM modulator 5 mM ALA + 20 mM modulator 30 mM modulator 5 mM ALA + 30 mM modulator Correlation coefficient Level of s i g n i f i c a n c e

Treatment

0 0 0 0 0 0 0 0

0 40 0 43 0 60 0 70

Soybean Cucumber

Photodynamic damage (*)

DV

MV

DV

0.00 -0.67 -0.90 -0:60 -1.19 -0.98 0.06 1.30

-

0.00 0.83 0.97 1.70 1.36 0.87 0.88 0.43

-

C o n t i n u e d on next page

0.00 4.62 3.13 15.34 •3:48 15.73 3.67 21.96 0.907 1$

Exogenous ALA-induced t e t r a p y r r o l e accumulation (nmoles per 100 mg p r o t e i n )

0.00 9.69 3.99 21.24 4.27 22.33 3.49 32.89 0.945 0.1$

MV

Pchlide

Cucumber

MPE

Response o f Cucumber, a DDV/LDV P l a n t S p e c i e s and o f Soybean, a DMV/LDV P l a n t S p e c i e s to Enhancers o f ALA C o n v e r s i o n t o MV P r o t o c h l o r o p h y l l i d e

Treatment c o n d i t i o n s , a b b r e v i a t i o n s and d e f i n i t i o n s a r e as i n T a b l e I . Adapted from ( 2 7 ) .

Table I I .

I

S3-

!

5

m CD m N m H > r

to to

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

picolinic acid

Chlorophyll biosynthesis modulator

Solvent only 5 mM ALA 10 mM modulator 5 mM ALA + 10 mM modulator 20 mM modulator

Treatment

0 3 3 6 3

0 34 0 71 0

Soybean Cucumber

Photodynamic damage «)

0.00 12.05 2.92 16.87 11.08

MV

DV

MV

MPE

DV

0.00 4.03 4.20 12.65 6.20

0.00 -0.15 -0.18 1.76 -0.33

0.00 -0.51 -0.69 -0.59 0.70

Exogenous ALA-induced tetrapyrrole accumulation (nmoles per 100 mg protein)

Pchlide

Cucumber

Table I I . Continued. Response o f Cucumber, a DDV/LDV P l a n t S p e c i e s , and of Soybean, a DMV/LDV P l a n t S p e c i e s , t o Enhancers of ALA C o n v e r s i o n t o MV P r o t o c h l o r o p h y l 1 i d e

5 m

m H n

o

m

1

H

X

r 0

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

,2'-dipyridyl disulfide

5 mM ALA + 20 mM modulator 30 mM modulator 5 mM ALA + 30 mM modulator Correlation coefficient Level of s i g n i f i c a n c e Solvent only 5 mM ALA 10 mM modulator 5 mM ALA + 10 mM modulator 20 mM modulator 5 mM ALA + 20 mM modulator 30 mM modulator 5 mM ALA + 30 mM modulator Correlation coefficient Level of s i g n i f i c a n c e 0 12 8 16 17 19 15 21

12 6 21

0 80 0 70 0 70 0 73

80 10 90 39.87 2.68 21 .47 0.828 5t 0.00 11.94 -2.94 18.08 -2.73 17.32 6.14 41.80 0.779 5%

17.35 2.35 9.06 0.811 5% 0.00 0.19 -0.96 1.96 0.31 3.70 1.89 4.87 0.598 20*

-

2.06 0.27 0.92 0.47 -0.16 0.28 -0.01 0.42 0.20 -1 .01 0.18 -0.43 0.75 1.07

-

0.00

0.00

-

0.55 0.69 3.99

-0.87 -0.72 -0.92

316

LIGHT-ACTIVATED PESTICIDES

o f growth. These r e s u l t s i n d i c a t e d t h a t t h e a c c u m u l a t i o n o f DV MP(E) by a p l a n t was l i k e l y t o cause photodynamic damage, i r r e s p e c t i v e o f t h e g r e e n i n g group t o which t h e p l a n t b e l o n g e d . O r i g i n o f t h e D i f f e r e n t i a l Photodynamic S u s c e p t i b i l i t y o f V a r i o u s P l a n t S p e c i e s t o ALA-Dependent T e t r a p y r r o l e A c c u m u l a t i o n : A Working H y p o t h e s i s . On t h e b a s i s o f t h e above, a l b e i t l i m i t e d , o b s e r v a t i o n s we now propose t h e f o l l o w i n g w o r k i n g h y p o t h e s i s : ( a ) t h a t P c h l i d e i s t h e most u b i q u i t o u s o f t h e damage-causing p h o t o dynamic t e t r a p y r r o l e s t h a t accumulate as a consequence o f ALA-based t r e a t m e n t s , (b) t h a t DDV/LDV p l a n t s p e c i e s a r e l i k e l y t o be more p h o t o d y n a m i c a l l y s u s c e p t i b l e t o ALA-based dark t r e a t m e n t s t h a t l e a d t o MV P c h l i d e a c c u m u l a t i o n , than t o those t h a t l e a d t o DV P c h l i d e a c c u m u l a t i o n . However, i t remains t o be determined whether t h i s i s due t o d i f f e r e n c e s i n t h e photodynamic damage-causing p o t e n t i a l between e q u i m o l a r amounts o f MV and DV P c h l i d e o r whether i t i s due to other f a c t o r s , ( c ) c a l l y more s u s c e p t i b l e t i o n , and (d) t h a t both DDV/LDV and DMV/LDV p l a n t s p e c i e s a r e h i g h l y s u s c e p t i b l e t o DV MP(E) a c c u m u l a t i o n . As was a l r e a d y p o i n t e d o u t , t h e p r e m i s e s o f t h i s h y p o t h e s i s are l i k e l y t o be expanded and/or r e f i n e d i n o r d e r t o accommodate a d d i t i o n a l o b s e r v a t i o n s d e r i v e d from a d d i t i o n a l photodynamic s u s c e p t i b i l i t y s t u d i e s o f t h e f o u r g r e e n i n g groups o f p l a n t s . Fur thermore, i t would be very d e s i r a b l e t o determine t h e r e a s o n why DDV/LDV p l a n t s p e c i e s appear t o be more s u s c e p t i b l e t o ALA-depen dent MV P c h l i d e d a r k - a c c u m u l a t i o n w h i l e DMV/LDV p l a n t s p e c i e s appear t o be more s u s c e p t i b l e t o DV t e t r a p y r r o l e d a r k - a c c u m u l a t i o n . M o d u l a t i o n o f AlA-dependent T e t r a p y r r o l e A c c u m u l a t i o n and Concommltant M o d u l a t i o n o f Photodynamic Damage by C h l o r o p h y l l Biosynthesis Modulators The o b s e r v a t i o n t h a t t h e photodynamic s u s c e p t i b i l i t y o f a p l a n t s p e c i e s depended on t h e g r e e n i n g group o f t h e p a r t i c u l a r p l a n t s p e c i e s a s w e l l as on t h e n a t u r e o f t h e accumulated t e t r a p y r r o l e s had o b v i o u s b i o t e c h n o l o g i c a l i m p l i c a t i o n s . I t suggested t h a t c h e m i c a l s t h a t may be a b l e t o induce A L A - t r e a t e d p l a n t s , b e l o n g i n g t o a c e r t a i n g r e e n i n g group, t o accumulate t h e "wrong" t y p e o f MV or DV t e t r a p y r r o l e , w h i l e i n d u c i n g o t h e r p l a n t s p e c i e s , b e l o n g i n g t o o t h e r g r e e n i n g groups, t o accumulate t h e " r i g h t " type o f MV o r DV t e t r a p y r r o l e may a c t as photodynamic h e r b i c i d e m o d u l a t o r s . I n o t h e r words, such c h e m i c a l s when used i n c o n j u n c t i o n w i t h ALA had the p o t e n t i a l t o expand t h e ALA h e r b i c i d e i n t o a h i g h l y s e l e c t i v e system o f photodynamic h e r b i c i d e s . With t h i s i n mind we undertook a l i t e r a t u r e s e a r c h f o r chemi c a l s and b i o c h e m i c a l s known t o a f f e c t i n a g e n e r a l way, C h i and P c h l f o r m a t i o n ( 4 4 - 4 6 ) . We then determined t h e s p e c i f i c e f f e c t o f t h e s e c h e m i c a l s on t h e v a r i o u s C h i a b i o s y n t h e t i c r o u t e s d e s c r i b e d i n F i g . 2. This research e f f o r t r e s u l t e d i n the i d e n t i f i c a t i o n o f a t o t a l o f 13 c h e m i c a l s which a c t e d i n c o n c e r t w i t h ALA and which were c a p a b l e o f m o d u l a t i n g t h e C h i a b i o s y n t h e t i c pathway. These chemi c a l s were, t h e r e f o r e , d e s i g n a t e d c o l l e c t i v e l y as C h i a b i o s y n t h e s i s

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

22.

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317

modulators. They were c l a s s i f i e d i n t o t h r e e major groups depending on t h e i r mode o f a c t i o n . One group c o n s i s t e d o f enhancers o f ALA c o n v e r s i o n t o t e t r a p y r r o l e s . Another group c o n s i s t e d o f i n d u c e r s o f ALA b i o s y n t h e s i s and o f t e t r a p y r r o l e a c c u m u l a t i o n w h i l e a t h i r d group c o n s i s t e d o f i n h i b i t o r s o f MV p r o t o c h l o r o p h y l l i d e accumulation. The e f f e c t o f these v a r i o u s groups o f C h i a b i o s y n t h e s i s modulators on t h e C h i a b i o s y n t h e t i c pathway and on induced photo dynamic damage i s d e s c r i b e d below. Enhancers o f ALA C o n v e r s i o n t o T e t r a p y r r o l e s . To q u a l i f y as an enhancer o f ALA c o n v e r s i o n t o a p a r t i c u l a r MV o r DV t e t r a p y r r o l e i t was c o n s i d e r e d t h a t : (a) a p a r t i c u l a r C h i b i o s y n t h e s i s modulator s h o u l d n o t r e s u l t I n a s i g n i f i c a n t a c c u m u l a t i o n o f t h e MV o r DV t e t r a p y r r o l e i n q u e s t i o n , when a p p l i e d t o a p l a n t i n t h e absence o f exogenous ALA, but (b) a t c e r t a i n c o n c e n t r a t i o n s o f t h e modulator, when t h e l a t t e r i s use enhance t h e dark t e t r a p y r r o l t h a t p a r t i c u l a r MV o r D t e t r a p y r r o l e , beyon c o n t r o l . A s i g n i f i c a n t accumulation o f a p a r t i c u l a r t e t r a p y r r o l e was i n t u r n d e f i n e d a r b i t r a r i l y as an amount o f t h a t t e t r a p y r r o l e t h a t approached o r exceeded t h e n e t d a r k - c o n v e r s i o n r a t e o f a 5 mM exogenous ALA t r e a t m e n t i n t o t h a t t e t r a p y r r o l e . Enhancers o f ALA c o n v e r s i o n t o t e t r a p y r r o l e s were observed t o f a l l i n t o two d i s t i n c t groups namely ( a ) enhancers o f ALA c o n v e r s i o n t o MV P c h l i d e and (b) enhancers o f ALA c o n v e r s i o n t o DV P c h l i d e . These two subgroups o f enhancers w i l l now be d i s c u s s e d separately. Enhancers o f ALA C o n v e r s i o n t o MV P c h l i d e . 2 - P y r i d i n e a l d e h y d e , p i c o l i n i c a c i d , 2 , 2 - d i p y r i d y l d i s u l f i d e ( T a b l e I I ) and 2 - p y r i d i n e a l d o x i m e , t h e l a t t e r i n t h e h i g h e r c o n c e n t r a t i o n range ( T a b l e I , A) were found t o enhance p r e f e r e n t i a l l y t h e d a r k - c o n v e r s i o n o f exo genous ALA t o MV P c h l i d e i n DDV/LDV p l a n t s p e c i e s such as cucumber. I t s h o u l d be emphasized, however, t h a t a l t h o u g h t h e s e compounds enhanced p r e f e r e n t i a l l y t h e d a r k - c o n v e r s i o n o f ALA t o MV P c h l i d e , some o f them a l s o enhanced s i g n i f i c a n t l y , but t o a l e s s e r e x t e n t , the d a r k - c o n v e r s i o n o f exogenous ALA t o DV P c h l i d e . I n DDV/LDV p l a n t s p e c i e s such as cucumber, a h i g h e r c o r r e l a t i o n was observed between photodynamic damage and t h e dark-accumula t i o n o f MV P c h l i d e , than between photodynamic damage and t h e accumu l a t i o n o f DV P c h l i d e . No s i g n i f i c a n t a c c u m u l a t i o n o f e i t h e r MV o r DV M g - p r o t o p o r p h y r i n s was observed (Table I I ) . Treatment o f soybean w i t h t h e s e same enhancers o f exogenous ALA c o n v e r s i o n t o MV P c h l i d e r e s u l t e d i n m i n i m a l o r no photodynamic damage ( T a b l e I I ) . T h i s i s f u l l y c o m p a t i b l e w i t h t h e proposed d i f f e r e n t i a l s u s c e p t i b i l i t y h y p o t h e s i s . P a r t i c u l a r l y i f soybean, a DMV/LDV p l a n t s p e c i e s r e a c t e d t o treatment w i t h ALA and 2 - p y r i d i n e aldehyde, p i c o l i n i c a c i d , 2 , 2 ' - d i p y r i d y l d i s u l f i d e o r 2 - p y r i d i n e a l d o x i m e , as d i d cucumber a DDV/LDV p l a n t s p e c i e s , by a c c u m u l a t i n g MV P c h l i d e . T h i s q u e s t i o n i s p r e s e n t l y under i n v e s t i g a t i o n . ,

Enhancers o f ALA C o n v e r s i o n t o DV P c h l i d e . 4,4»-dipyridyl, 2,2'd i p y r i d y l amine and p h e n a n t h r i d i n e were observed t o f a l l i n t o t h i s

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

318

LIGHT-ACTIVATED PESTICIDES

group o f C h i b i o s y n t h e s i s m o d u l a t o r s . At t h e h i g h e r - c o n c e n t r a t i o n range, they enhanced p r e f e r e n t i a l l y t h e dark c o n v e r s i o n o f exo genous ALA t o DV P c h l i d e i n t r e a t e d cucumber s e e d l i n g s . S i n c e cucumber i s a DDV/LDV p l a n t s p e c i e s , i t was l e s s p h o t o d y n a m i c a l l y s e n s i t i v e t o f l u c t u a t i o n s i n i t s DV P c h l i d e than t o f l u c t u a t i o n s i n i t s MV P c h l i d e c o n t e n t . T h i s i n t u r n was r e f l e c t e d by a b e t t e r c o r r e l a t i o n between photodynamic damage and MV P c h l i d e a c c u m u l a t i o n t h a n between photodynamic damage and DV P c h l i d e a c c u m u l a t i o n ( T a b l e III). No s i g n i f i c a n t a c c u m u l a t i o n o f e i t h e r MV o r DV Mg-protoporp h y r i n s was o b s e r v e d . I n c o n t r a s t t o cucumber, d a r k - t r e a t m e n t o f soybean w i t h ALA t o g e t h e r w i t h t h e f o r e m e n t i o n e d enhancers o f ALA c o n v e r s i o n t o DV P c h l i d e , r e s u l t e d i n e x t e n s i v e photodynamic damage ( T a b l e I I I ) . T h i s was t h e e x p e c t e d phenomenology i f t h e dark t r e a t m e n t o f soy bean, a DMV/LDV p l a n t s p e c i e s , w i t h ALA and 4 , 4 ' - d i p y r i d y l , 2,2'd i p y r i d y l amine o r p h e n a n t h r i d i n e had t r i g g e r e d an enhancement o r an i n d u c t i o n o f DV t e t r a p y r r o l p r e s e n t l y under i n v e s t i g a t i o n I n d u c e r s o f T e t r a p y r r o l e A c c u m u l a t i o n . To q u a l i f y a s an i n d u c e r o f t e t r a p y r r o l e a c c u m u l a t i o n , i t was c o n s i d e r e d t h a t a p a r t i c u l a r C h i b i o s y n t h e s i s modulator s h o u l d , a t c e r t a i n c o n c e n t r a t i o n s , r e s u l t i n a s i g n i f i c a n t a c c u m u l a t i o n o f a p a r t i c u l a r MV o r DV t e t r a p y r r o l e , when a p p l i e d t o a p l a n t i n t h e absence o f exogenous ALA. Here a g a i n , s i g n i f i c a n t a c c u m u l a t i o n o f a p a r t i c u l a r t e t r a p y r r o l e was a r b i t r a r i l y d e f i n e d as an amount o f t h a t t e t r a p y r r o l e t h a t a p proaches o r exceeds t h e n e t d a r k - c o n v e r s i o n r a t e o f a 5 mM exogen ous ALA t r e a t m e n t i n t o t h a t t e t r a p y r r o l e . Furthermore, a t c e r t a i n c o n c e n t r a t i o n s o f t h e i n d u c e r , t h e l a t t e r , i n c o m b i n a t i o n w i t h ALA, should r e s u l t i n the accumulation o f higher l e v e l s o f the p a r t i c u l a r MV o r DV t e t r a p y r r o l e than when ALA o r t h e i n d u c e r a r e a p p l i e d to t h e p l a n t s e p a r a t e l y . 1,1O-phenanthroline ( i . e . O - p h e n a n t h r o l i n e ) ( T a b l e I , D) and 2 , 2 - d i p y r i d y l ( T a b l e I V ) were observed t o a c t p r e f e r e n t i a l l y as i n d u c e r s o f DV M g - p r o t o p o r p h y r i n + DV M g - p r o t o p o r p h y r i n monoester [DV MP(E)] a c c u m u l a t i o n . I t s h o u l d be noted t h a t w h i l e 1,1O-phenan t h r o l i n e p r e f e r e n t i a l l y induced t h e b i o s y n t h e s i s and a c c u m u l a t i o n of DV MP(E), i t a l s o i n d u c e d , t o a l e s s e r e x t e n t , t h e a c c u m u l a t i o n o f DV P c h l i d e ( T a b l e I D). 2 , 2 • - d i p y r i d y l ( T a b l e I V ) d i d n o t e x h i b i t t h i s p r o p e r t y . I n cucumber t h e h i g h e s t c o r r e l a t i o n was observed between DV MP(E) a c c u m u l a t i o n and photodynamic damage ( T a b l e s I , D and I V ) . Soybean a DMV/LDV p l a n t s p e c i e s was e q u a l l y s u s c e p t i b l e t o t r e a t m e n t w i t h 2 , 2 - d i p y r i d y l ( T a b l e I V ) and t o 1,1O-phenanthroline ( d a t a n o t shown). T h i s i n t u r n was c o m p a t i b l e w i t h t h e proposed mode o f a c t i o n h y p o t h e s i s . I n v e s t i g a t i o n s o f t h e q u a n t i t a t i v e r e l a t i o n s h i p s between t h e i n d u c t i o n o f s p e c i f i c t e t r a p y r r o l e accumula t i o n and t h e i n c i d e n c e o f photodynamic damage i n DMV/LDV p l a n t s p e c i e s such a s soybean a r e i n p r o g r e s s . f

f

I n h i b i t o r s o f MV P r o t o c h l o r o p h y l l l d e A c c u m u l a t i o n . To q u a l i f y a s an i n h i b i t o r o f MV P c h l i d e a c c u m u l a t i o n , i t was c o n s i d e r e d t h a t a p a r t i c u l a r C h i b i o s y n t h e s i s modulator (a) when used a l o n e , s h o u l d r e s u l t i n t h e i n h i b i t i o n o f MV P c h l i d e a c c u m u l a t i o n , i n comparison

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

4,4'-Dipyridyl

Chlorophyll biosynthesis modulator

Solvent only 5 mM ALA 10 mM modulator 5 mM ALA + 10 mM modulator 20 mM modulator

Treatment

0 10 15 50 58

0 20 0 15 0

Soybean Cucumber

Photodynamic damage

0.00 19.66 -1.82 11.09 -1.20

MV

DV

MV

MPE

DV

0.00 0.00 4.06 -1.09 -1.90 -1.09 4.44 -0.31 -0.44 -0.56 Continued on next

0.00 -0.82 -1.02 -0.69 -0.85 page

Exogenous ALA-induced t e t r a p y r r o l e accumulation (nmoles per 100 mg p r o t e i n )

Pchlide

Cucumber

Response o f Cucumber, a DDV/LDV P l a n t S p e c i e s and o f Soybean, a DMV/LDV P l a n t S p e c i e s t o Enhancers o f ALA C o n v e r s i o n t o DV P r o t o c h l o r o p h y l l i d e

Treatment a b b r e v i a t i o n s and d e f i n i t i o n s a r e as i n T a b l e I . Adapted from ( 2 7 ) .

Table I I I .

I

5

m H > r

N

2

DO

m

70

to

lO

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chlorophyll biosynthesis modulator

5 mM ALA + 20 mM modulator 30 mM modulator 5 mM ALA + 30 mM modulator Correlation coefficient Level of s i g n i f i c a n c e Solvent only 5 mM ALA 10 mM modulator 5 mM ALA + 10 mM modulator

Treatment

0 15 0 36

76 85 93

Soybean

25

-

0 25

-

75 0 25

Cucumber

Photodynamic damage

•

• -

15.37

DV

MV

MPE

DV

9.91

-

22.68 9.21 34.54 0.647 10% 0.00 3.87

-1.71

-

-

0.00 -0.69

0.31 0.73 -0.08

-0.81

0.00 -0.87

-

-0.09 -0.81 -0.39

Exogenous ALA-induced t e t r a p y r r o l e accumulation (nmoles per 100 mg p r o t e i n )

20.69 -1.34 22.56 0.760 5% 0.00 16.23

MV

Pchlide

Cucumber

Table I I I . C o n t i n u e d . Response of Cucumber, a DDV/LDV P l a n t S p e c i e s , and of Soybean, a DMV/LDV P l a n t S p e c i e s , t o Enhancers of ALA C o n v e r s i o n t o DV P r o t o c h l o r o p h y l 1 i d e

a m

n

C/3

rn H

m a -v

1

ft

x

r 0

O

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

phenanthridine

2,2'-Dipyridyl amine

20 mM modulator 5 mM ALA + 20 mM modulator 30 mM modulator 5 mM ALA + 30 mM modulator Correlation coefficient Level of s i g n i f i c a n c e Solvent only 5 mM ALA 10 mM modulator 5 mM ALA + 10 mM modulator 20 mM modulator 5 mM ALA + 20 mM modulator 30 mM modulator 5 mM ALA + 30 mM modulator Correlation coefficient Level of significance 0 0 84 87 92 94 100 100

6 64 9 82

-

0 75 10 80 15 70 10 80

-

0 30 0 15

3.70 20.19 0.94 15.48 0.962 0.1$ 0.00 44.20 12.25 36.11 8.98 49.60 5.46 57.05 0.955 0.1$ 0.53 20.67 0.09 10.80 0.81 5$ 0.00 16.82 5.80 17.70 6.67 29.79 3.33 72.84 0.706 10$

-

-

-

0.00 0.86 0.27 0.78 0.01 0.31 0.56 1.87

0.43 -1.97 0.60 -0.80

-

-

0.00 0.35 0.12 0.24 -0.01 -0.04 0.38 1.76

-0.36 -1.01 -0.62 -0.92

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

2,2'-dipyridyl

Chlorophyll biosynthesis modulator

Solvent only 5 mM ALA 10 mM modulator 5 mM ALA + 10 mM modulator 20 mM modulator 5 mM ALA + 20 mM modulator 30 mM modulator 5 mM ALA + 30 mM modulator Correlation coefficient Level o f s i g n i f i c a n c e

Treatment

0 0 6 34 46 53 80 90

0 45 0 25 0 90 25 100

Soybean Cucumber

Photodynamic damage

DV

MV

Cucumber

MPE

DV

0.00 46.36 7.69 19.60 -4.76 37.20 -3.96 29.73 0.7415 10*

0.00 1.65 -0.86 0.73 1.39 5.34 6.07 14.30 0.814 5%

0.00 -0.58 -0.09 4.50 15.29 70.39 15.32 160.04 0.846 5%

Exogenous ALA-induced t e t r a p y r r o l e accumulation (nmoles per 100 mg p r o t e i n )

0.00 100.16 31.45 75.40 2.94 28.94 8.73 45.16 0.329 n.s.

MV

Pchlide

Treatments, a b b r e v i a t i o n s and d e f i n i t i o n s a r e a s i n T a b l e I . Adapted from ( 2 7 ) .

Table I V . Response o f Cucumber, a DDV/LDV P l a n t S p e c i e s and o f Soybean, a DMV/LDV P l a n t S p e c i e s t o I n d u c e r s o f DV M g - p r o t o p o r p h y r i n a c c u m u l a t i o n

UJ

rn

5

n

m a m C/J H

1

ft

r 0

to

22.

REBEIZ ET AL.

Photodynamic

Herbicides

323

t o t h e u n t r e a t e d c o n t r o l s , and/or (b) when used i n c o n j u n c t i o n w i t h ALA, i t s h o u l d r e s u l t i n t h e i n h i b i t i o n o f MV P c h l i d e a c c u m u l a t i o n i n comparison t o t h e A L A - t r e a t e d c o n t r o l . 4 , 7 - p h e n a n t h r o l i n e , 2 , 3 - d i p h y r i d y l and 2 , 4 - d i p y r i d y l (Table V) as w e l l as 1 , 7 - p h e n a n t h r o l i n e (Table I C) f a l l i n t o t h i s group o f C h i b i o s y n t h e s i s m o d u l a t o r s . I n most cases so f a r I n v e s t i g a t e d , when t h e i n h i b i t o r was used j o i n t l y w i t h ALA, e s p e c i a l l y a t t h e h i g h e r c o n c e n t r a t i o n l e v e l s o f i n h i b i t o r , t h e i n h i b i t i o n o f MV P c h l i d e d a r k - a c c u m u l a t i o n was accompanied by an enhancement o f DV P c h l i d e a c c u m u l a t i o n , i n comparison t o t h e A L A - t r e a t e d c o n t r o l (Table V ) . M g - p r o t o p o r p h y r i n a c c u m u l a t i o n was n o t o b s e r v e d . I n cucumber, a DDV/LDV p l a n t s p e c i e s , i n h i b i t o r - i n d u c e d photo dynamic damage over and beyond t h e A L A - t r e a t e d c o n t r o l s was e i t h e r minimal (4,7-phenanthroline, 2 , 3 - d i p y r i d y l i n Table V and 1,7p h e n a n t h r o l i n e i n T a b l e I C) or was absent ( 2 , 4 - d i p y r i d y l i n T a b l e V ) . However, i n soybean a DMV/LDV p l a n t s p e c i e s , t h e s e same ALA + i n h i b i t o r treatments r e s u l t e and beyond t h e A L A - t r e a t e i n t u r n f u l l y c o m p a t i b l e w i t h t h e proposed mode o f a c t i o n hypothesis. Epilogue The r e s e a r c h e f f o r t d e s c r i b e d i n t h i s work has a l r e a d y l e d t o t h e development o f photodynamic h e r b i c i d e f o r m u l a t i o n c a p a b l e o f con t r o l l i n g broad l e a f weeds i n Kentucky b l u e g r a s s , under f i e l d c o n d i t i o n s (47) and i n c o n t r o l l i n g s e v e r a l monocot and d i c o t weed s p e c i e s i n c o r n and soybean under greenhouse c o n d i t i o n s . I n sum mary such an e f f o r t has i n v o l v e d (a) t h e c l a s s i f i c a t i o n o f the p l a n t s p e c i e s t o be d e s t r o y e d and t h o s e t o be saved i n t o t h e i r r e s p e c t i v e g r e e n i n g groups, (b) s e l e c t i o n o f one o r more C h i b i o s y n t h e s i s m o d u l a t o r s t o a c t j o i n t l y w i t h ALA and t o Induce t h e u n d e s i r a b l e weeds t o accumulate u n d e s i r a b l e t e t r a p y r r o l e s t h a t do not b e l o n g t o a f u n c t i o n a l b i o s y n t h e t i c r o u t e , ( c ) development o f a f i e l d s o l v e n t system c a p a b l e o f d e l i v e r i n g t h e ALA and t h e C h i b i o s y n t h e s i s m o d u l a t o r ( s ) t o t h e c h l o r o p l a s t , where ALA i s c o n v e r t e d t o t e t r a p y r r o l e s and f i n a l l y (d) t e s t i n g t h e developed s o l v e n t system under t h e f i e l d c o n d i t i o n s f o r which i t had been designed (47). Because o f t h e p o s s i b i l i t y o f combining i n d i v i d u a l members o f the f o u r c l a s s e s o f C h i b i o s y n t h e s i s m o d u l a t o r s and ALA, f i v e , f o u r , t h r e e o r two a t a t i m e , i t i s p o s s i b l e t o d e s i g n a v e r y l a r g e number o f u s e f u l h e r b i c i d e s . For example w i t h t h e 13 C h i b i o s y n t h e s i s m o d u l a t o r s d e s c r i b e d i n t h i s work, i t i s a l r e a d y p o s s i b l e t o d e s i g n 3458 d i f f e r e n t h e r b i c i d a l m i x t u r e s . On t h e o t h e r hand t h e d i s c o v e r y o f one o r two a d d i t i o n a l C h i b i o s y n t h e s i s m o d u l a t o r s has the p o t e n t i a l o f r e s u l t i n g i n 1470 and 2410 a d d i t i o n a l h e r b i c i d e s respectively. Acknowledgments T h i s work was s u p p o r t e d by N a t i o n a l S c i e n c e F o u n d a t i o n Grant DMB 85-07217, by funds from t h e I l l i n o i s A g r i c u l t u r a l Experiment S t a t i o n and by t h e John P. T r e b e l l a s P h o t o b i o t e c h nology Research Endowment t o C.A.R.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

4,7-phenan throline

Chlorophyll biosynthesis modulator

Solvent only 5 mM ALA 10 mM modulator 5 mM ALA + 10 mM modulator 20 mM modulator

Treatment

0 3 93 94 100

0 60 3 45 21

Soybean Cucumber

Photodynamic damage «}

0.00 41.65 -0.24 19.18 -5.33

MV

DV

MV

MPE

DV

0.00 8.04 2.66 16.00 3.09

0.00 0.92 0.20 1.05 0.99

0.00 0.91 0.34 0.19 -0.95

Exogenous ALA-induced t e t r a p y r r o l e accumulation (nmoles per 100 mg p r o t e i n )

Pchlide

Cucumber

Response o f Cucumber a DDV/LDV P l a n t S p e c i e s and o f Soybean, a DMV/LDV P l a n t S p e c i e s t o I n h i b i t o r s o f MV P r o t o c h l o r o p h y l l i d e A c c u m u l a t i o n

Treatments, a b b r e v i a t i o n s and d e f i n i t i o n s a r e as i n T a b l e I . Adapted from ( 2 7 ) .

Table V.

r

3 m

n

m

T3

m D

I

H

X

5

to

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

2,3-dipyridyl

5 mM ALA + 20 mM modulator 30 mM modulator 5 mM ALA + 30 mM modulator Correlation coefficient Level of s i g n i f i c a n c e Solvent only 5 mM ALA 10 mM modulator 5 mM ALA + 10 mM modulator 20 mM modulator 5 mM ALA + 20 mM modulator 30 mM modulator 5 mM ALA + 30 mM modulator Correlation coefficient Level of s i g n i f i c a n c e 0 8 28 35 40 45 65 70

94 100 97

-

0 49 0 25 0 28 0 40

-

56 48 88

12.08 0.74 -4.58 0.299 n.s. 0.00 65.42 6.08 26.16 -1.01 41.56 8.46 19.50 0.869 5%

-

-

0.00 -0.25 -0.22 0.70 1.47 -0.79 -0.14 0.63

1.38 0.86 1.21

-

1.48 -0.31 0.62 3.98 1.47 2.01

6.27

0.00

—

-0.45 0.19 -0.56

C o n t i n u e d on next page

18.85 14.60 25.70 0.889 1* 0.00 30.80 5.55 31.26 6.82 58.53 14.59 38.75 0.763 10*

to

§

5

N

m H > r

m DO m

70

to to

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

2,4-dipyridyl

Chlorophyll biosynthesis modulator

10 mM modulator 5 mM ALA + 10 mM modulator 20 mM modulator 5 mM ALA + 20 mM modulator 30 mM modulator 5 mM ALA + 30 mM modulator Correlation coefficient Level o f significance

5 mM ALA

Solvent only

Treatment

47 60

40

20

13 25

3 8

-

0 8 0 5

-

0 35 0

Soybean Cucumber

Photodynamic damage «}

8.15

5%

3.44 0.927

-5.71

9.28

-

-5.05

n.s.

0.227

-

-0.12 11.63 4.15 26.54

-0.63

0.00

0.00 -3.32

MV

MPE

DV

0.00

-0.46 0.57 0.82 0.30

-

-0.16 0.57 0.34

-

-

-1.12 -1.03 1.32

-

0.93 0.32

0.00

Exogenous ALA-induced t e t r a p y r r o l e accumulation (nmoles per 100 mg p r o t e i n )

DV

Cucumber

19.65

MV

Pchlide

Table V. C o n t i n u e d . Response o f Cucumber, a DDV/LDV P l a n t S p e c i e s , and of Soybean, a DMV/LDV P l a n t S p e c i e s , t o I n h i b i t o r s o f MV P r o t o c h l o r o p h y 1 1 i d e A c c u m u l a t i o n

m

5

m a m H n

1

H

ft

r 0

to

22.

REBEIZ ET AL.

Photodynamic Herbicides

327

Legend o f Symbols. P c h l i d e : p r o t o c h l o r o p h y l l i d e ; P r o t o : protopor p h y r i n I X ; Mg-proto; Mg p r o t o p o r p h y r i n I X ; MPE: M g - p r o t o p o r p h y r i n monoester; MP ( E ) : a m i x t u r e o f Mg-Proto and MPE; C h i : c h l o r o p h y l l ; monocot: monocotyledonous p l a n t ; d i c o t : dicotyledonous p l a n t ; MV: m o n o v i n y l ; DV: d i v i n y l ; C h l i d e : c h l o r o p h y l l i d e ; P r o t o gen: p r o t o p o r p h y r i n o g e n ; A l k . E: a l k y l e s t e r ; P c h l : protochloro p h y l l i d e e s t e r ; P c h l ( i d e ) : p c h l i d e + P c h l ; DPy: 2 , 2 - d i p y r i d y l . 1

Literature Cited 1.

Rebeiz, C. A.; Montazer-Zouhoor, A.; Hopen, H. J . ; Wu, S. M. Enzyme Microb. Tecnol. 1984, 6, 390-401. 2. Hopf, F. R.; Whitten, D. G. In The Porphyrins; Dolphin, D., Ed.; Academic: New York, 1978; Vol. 2, pp 161-195. 3. Foote, C. S. In Porphyrin Localization and Treatment of Tumors; Alan R. L i s s : New York, 1984; pp 3-18. 4. Mattheis, J . R.; Rebeiz 4022-4024. 5. Mattheis, J . R.; Rebeiz, C. A. J . B i o l . Chem. 1977, 252, 8347-8349. 6. Mattheis, J . R.; Rebeiz, C. A. Photochem. Photobiol. 1978, 28, 55-60. 7. Hougen, C. L.; Meller E.; Gassman, M. L. Plant Science Letters 1982, 24, 289-294. 8. Rebeiz, C. A.; Mattheis, J . R.; Smith, B. B.; Rebeiz C. C.; Dayton, D. F. Arch. Biochem. Biophys. 1975, 171, 549-567. 9. Smith, B. B.; Rebeiz, C. A. Photochem. Photobiol. 1977, 26, 527-532. 10. Bazzaz, M. B.; Rebeiz, C. A. Photochem. Photobiol. 1979, 30, 709-721. 11. Rebeiz, C. A.; Daniell, H.; Mattheis, J . R. In 4th Symposium on Biotechnology i n Energy Production and Conservation; Scot, C. D., Ed.; John Wiley: New York, 1982; pp 413-439. 12. S l s l e r , E. C.; Klein, W. Physiol. Plant. 1963, 16, 315-322. 13. Rebeiz, C. A.; Abou Haidar, M.; Yaghi, M.; Castelfranco, P. A. Plant Physiol. 1970, 46, 543-549. 14. Rebeiz, C. A.; Mattheis, J . R.; Smith, B. B.; Rebeiz, C. C.; Dayton, D. F. Arch. Biochem. Biophys. 1975, 166, 446-465. 15. Granick, S.; Mauzerall, D. In Metabolic Pathways; Greenberg, D. M., Ed.; Academic: New York, 1961; pp 525-615. 16. Rebeiz, C. A.; Castelfranco, P. A. Plant Physiol. 1973, 24, 129-172. 17. Lascelles, J . In Porphyrins and Related Compounds; Goodwin, T. W., Ed.; Academic: New York, 1968; pp 49-59. 18. Rebeiz, C. A.; Lascelles, J . In Photosynthesis: Energy Conversion by Plants and Bacteria; Govindjee, Ed.; Academic: New York, 1982; Vol. 1, pp 699-780. 19. Rebeiz, C. A.; Yaghi, M.; Abou-Haidar, M.; Castelfranco, P. A. Plant Physiol. 1970, 46, 57-63. 20. Daniell, H.; Rebeiz, C. A. Biochem. Biophys. Res. Commun. 1982, 104, 837-843. 21. Daniell, H.; Rebeiz, C. A. Biochem. Biophys. Res. Commun. 1982, 106, 466-470. 22. Daniell, H.; Rebeiz, C. A. Biotech. Bioeng. 1984, XXII, 481-487. 23. Granick; S. J . B i o l . Chem. 1950, 183, 713-730.

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

LIGHT-ACTIVATED PESTICIDES

328 24.

25.

26. 27.

28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38.

39. 40. 41. 42. 43.

44. 45. 46. 47.

Castelfranco, P. A.; Beale, S. I . In The Biochemistry of Plants; Hatch, M. D.; Boardman, N. K., Eds.; Academic: New York, 1981; Vol. 8, pp 375-421. Rebeiz, C. A.; Belanger, F. C.; McCarthy, C. A.; Freyssinet, G.; Duggan, J . X.; Wu, S. M.; Mattheis, J . R. In Photosynthesis. Chloroplast Development; Akoyunoglou, G., Ed.; Balaban International Science Services: Philadelphia, 1981; Vol. 5, pp 197-212. Rebeiz, C. A.; Wu, S. M.; Kuhadja, M.; D a n i e l l , H.; Perkins, E. J . Mol. C e l l . Biochem. 1983, 57, 97-125. Rebeiz, C. A.; Montazer-Zouhoor, A.; Mayasich, J. M.; Tripathy, B. C.; Wu, S. M.; Rebeiz, C. C. C r i t . Rev. Plant L e i . In press. McCarthy, S. A.; Belanger, F. C.; Rebeiz, C. A. Biochemistry 1981, 20, 5080-5087. Belanger, F. C.; Rebeiz, C. A. J . B i o l . Chem. 1982, 257, 1360-1371. Belanger, F. C.; Rebeiz 4875-4883. Belanger, F. C.; Rebeiz, C. A. Plant Sci. Lett. 1980, 18, 343-350. McCarthy, S. A.; Mattheis, J . R.; Rebeiz, C. A. Biochemistry 1982, 21, 242-247. Belanger, F. C.; Rebeiz, C. A. J . B i o l . Chem. 1980, 255, 1266-1272. Duggan, J . X.; Rebeiz, C. A. Plant S c i . Lett. 1982, 24, 27-37. Wu, S. M.; Rebeiz, C. A. Tetrahedron 1984, 40, 659-664. Belanger, F. C.; Duggan, J . X.; Rebeiz, C. A. J . B i o l . Chem. 1982, 257, 4849-4858. Duggan, J . X.; Rebeiz, C. A. Plant S c i. Lett. 1982, 27, 137-145. Rebeiz, C. A.; Tripathy, B. C.; Wu, S. M.; MontazerZouhoor, A.; Carey, E. E. In Regulation of Chloroplast D i f f e r e n t i a t i o n ; Akoyunoglou, G.; Senger, H., Eds.; Alan R. Liss: New York, 1986; pp 13-24. Tripathy, B. C.; Rebeiz, C. A. J . B i o l . Chem. 1986, 26 13556-13564. Tripathy, B. C.; Rebeiz, C. A. Anal. Biochem. 1985, 149, 43-61. Carey, E. E.; Tripathy, B. C.; Rebeiz, C. A. Plant Physiol 1985, 79, 1059-1063. Carey, E. E.; Rebeiz, C. A. Plant Physiol. 1985, 79, 1-6. Rebeiz, C. A.; Montazer-Zouhoor, A.; Rebeiz, C. C. In Thirty Eighth Illinois Custom Spray Operators Training Manual, University of Illinois Cooperative Extension Service, Ed.; Univ. I l l i n o i s Press: Urbana, IL, 1986; pp 91-93. Jones, O. T. G. Biochem. J . 1963, 88, 335-343. Duggan, J.; Gassman, M. Plant Physiol. 1974, 53, 206-215. Bednarick, D. P.; Hoober, J . K. Arch. Biochem. Biophys. 1985, 240, 369-379. Rebeiz, C. A.; Rebeiz, C. C.; Montazer-Zouhoor, A. American Lawn Applicator. 1987, In Press.

R E C E I V E D December29,1986

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Author Index Mayasich, J. M., 295 Montazer-Zouhoor, A., 295 Morand, P., 255 Nemec, Stan, 281 Patterson, R. S., 156 Philogene, B. J . R., 255 Pimprikar, G. D., 134 Pooler, John P., 109 Rebeiz, C. A., 295 Rebeiz, C. C , 295 Rodgers, Michael A. J . , 76 Samuels, Richard I . , 265 Scaiano, J . C , 255

Arnason, J . T., 255 Bennett, William J . , 176 Berenbaura, M. R. 206 Bindokas, Vitautas, 176 Champagne, Donald E., 231 Coign, Mary Jane, 134 Cooper, Geoffrey K. 241 Daub, Margaret E., 271 Dodge, Alan D., 265 Downum, Kelsey R. 281 Eickhoff, Thomas, 156 Feger, Mary B., 156 Foote, Christopher S., 22 Heitz, James R., I v i e , G. Wayne, 21 Kagan, Edgard D., Kagan, Isabelle A., 176 Kagan, Jacques, 176 Khan, Ahsan U., 58 Knox, J . Paul, 265 Koehier, P. G., 156 Lam, J . , 255 Lemke, Lisa A., 156 Maas, Jacqueline L., 176 Marchant, Y. Yoke, 168,241 f

f

f

Straight, , Sweeney, Susan A., 176 Towers, G. H. N e i l , 231 Tripathy, B. C., 295 Tuveson, R. W., 192 Valenzeno, Dennis Paul, 39 Weaver, Joseph E., 122 Werstiuk, N., 255 Wu, S. M., 295

Affiliation Index U.S. Department of Agriculture, 156,217,281 University of Aarhus, 255 University of Bath, 265 University of B r i t i s h Columbia, University of C a l i f o r n i a , 22 University of F l o r i d a , 156 University of I l l i n o i s , Chicago, 176 University of I l l i n o i s , Urbana, 192,206,295 University of Kansas Medical Center, 39 University of Texas at Austin, 76 University of Utah School of Medicine, 98 University of Utah, 98 West V i r g i n i a University, 122

ARCO Plant C e l l Research I n s t i t u t e , 168,241 Emory University School of Medicine, 109 F l o r i d a International University, 281 F l o r i d a State University, 58 Harvard University, 58 H i l t o n Davis Chemical Company, 156 McMaster University, 255 M i s s i s s i p p i State University, 1,134 National Research Council, Ottawa, 255 North Carolina State University, 271 Ottawa-Carleton I n s t i t u t e for Graduate Studies and Research i n Biology and Chemistry, 255

330

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

331

INDEX

Subject Index A Acetylcholinesterase dye-sensitized photooxidations vs. photodynamic action, 5 i n a c t i v a t i o n by photoactive p e s t i c i d e , 12 Acetylcholinesterase system, e f f e c t of photodynamic action, 139t,l40 Adaptive response, d e s c r i p t i o n , 193 Adulticides, house f l y c o n t r o l , 160-162 A f l a t o x i n s , structure and phototoxicity, 233 Alcohols, photodynamic e f f e c t s , 100 Amino acids, photodynamic e f f e c t s , 101-102 6-Aminolevulinic acid (ALA) choice as a herbicide, 297 photodynamic action on plants, 14 s e l e c t i v e h e r b i c i d a l e f f e c t , 297,300 6-Aminolevulinic acid dependent tetrapyrrole accumulation enhancers of ALA conversion to pchlide, 316 modulation and concomitant modulation of photodynamic damage, 316-317 photodynamic s u s c e p t i b i l i t y , 316 Angelicin structure, 10-11 t o x i c i t y , 10 Anthracene d e r i v a t i v e s , diagnostic trap f o r s i n g l e t oxygen, 30 Arginine, photodynamic e f f e c t s , 102

B Behavioral resistance to phototoxin, concealed feeding, 209,210t Biochemical components, e f f e c t by photodynamic action, 136-137 Biochemical resistance to phototoxins d e t o x i f i c a t i o n pathways, 213 quenching, 212,213t Biochemistry of photodynamic reactions c e l l s and organelles, 105-106 s o l u t i o n , 100-105 Biocides, photodecomposition, 168-173 B i o l o g i c a l membranes e f f e c t by photodynamic action, 137-139

B i o l o g i c a l membranes—Continued f l u i d mosaic model, 40,42f Biomolecules, photodynamic e f f e c t s , 104-105 Biosynthetic enzymes, s i n g l e t oxygen generation, 69 Biosynthetic pathway f o r chlorophylla, mechanism, 300-301,302f,303f B i o t i c and o v i c i d a l e f f e c t s , e f f e c t s of photosensitizer treatment 149-150,151f,152

C

C . ene-diyne-ene tetrahydropyranyl ethers, structure, 242,243f C acetylenes, b i o l o g i c a l l y active compounds, 242t Carbohydrates, photodynamic e f f e c t s , 100 Carotenoids, e f f e c t on cercosporin resistance, 275-276 C e l l u l a r photodynamic action, physiological e f f e c t s , 125-126 12

17

Cercospora species, future p e r s p e c t i v e s f o r c o n t r o l , 278

Cercosporin i s o l a t i o n , 272 membrane damage to plant c e l l s , 273-274 photoactivated t o x i c i t y , 272-273 photodynamic e f f e c t s , 13,33 photosensitizing a c t i v i t y , 272 phototoxicity, 236 resistance mechanisms, 274 role as a e r i a l pathogens, 271 s i n g l e t oxygen detection, 34 structure, 13,16,33,271-272 Characterization techniques of s i n g l e t oxygen chemical traps, 29-31 comparison to clean sources of s i n g l e t oxygen, 31-32 conductivity, 3 3 , 3 4 f D 0 e f f e c t , 31 i n h i b i t o r s , 31 luminescence, 32-33,34f transient absorption spectroscopy, 33,34f 2

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

332

LIGHT-ACTIVATED PESTICIDES

Chemical generation of s i n g l e t oxygen, triethylsilane-ozone reaction, 69,72 Chemical probes, determination of s i n g l e t oxygen, 79-80 Chemical traps, characterization of s i n g l e t oxygen, 29-31 Chloroperoxidase a c t i v i t y , 68 s i n g l e t oxygen generation, 68-69,70f Chlorophyll biosynthesis, target f o r h e r b i c i d a l action, 14 Chlorophylla, biosynthetic pathway, 300-301,302f,303f Cholesterol, diagnostic trap for s i n g l e t oxygen, 29 Cloned carotenoid genes of Escherichia c o l i , protection of c e l l against phototoxins, 201,203 Continuous wave e x c i t a t i o apparatus, 82,83f spectrum, 8lf,82

D

Delayed r e c t i f i e r , description, 116 Deoxyribonucleic acid repair systems adaptive response, 193 error-prone repair, 193 excision repair, 193 mismatch repair, 193 oxidative stress, 194 photoreactivation, 193 recombination repair, 193 Destruction of c e l l s , photosensitizers, 125 Developmental t o x i c i t y b i o t i c and o v i c i d a l e f f e c t s , 149-152 c h a r a c t e r i s t i c s , 141 delayed developmental periods, 145,147-149 morphological abnormalities, 141-145 D i e l e c t r i c constant b i o l o g i c a l dependent photodynamic suscepti b i l i t y , biochemical o r i g i n , 306-316 D i f f u s i o n across membranes, s i n g l e t oxygen, 51,52f,53 5,8-Dimethoxypsoralen, structure, 287 Dimethylstilbene, competition between type I and type I I photooxidation reaction, 27-28 Dimol luminescence e f f i c i e n c y , 26 s i n g l e t oxygen detection, 32 2,3-Diphenyldioxene, use as chemical probe, 80 Diphenylisobenzofuran, use as chemical trap, 80 1,1-Diphenylmethoxyethylene, competition between type I and type I I photooxidation reactions, 27

DpO e f f e c t , characterization of s i n g l e t oxygen, 32 Dark d i v i n y l / l i g h t d i v i n y l and dark monovinyl/light d i v i n y l plant species differences i n tetrapyrrole accumulation and photodynamic damage, 308,309-312t response to enhancers of ALA conversion, 308,313-315t enhancers of ALA 2 , 2 ' - D i p y r i d y l , p h o t o d y n a m i c a c t i o n on conversion to d i v i n y l p l a n t s , 14 protochlorophyHide, 318,319-3211 D i v i n y l - and monovinylpchlide inducers of divinylprotoporphyrin accumulation patterns, accumulation, 3l8,322t biosynthetic o r i g i n , 304-305 i n h i b i t o r s of monovinylDyes protochlorophyllide, 323,324-326t p h o t o s t a b i l i t y , 171-172 Dark d i v i n y l / l i g h t d i v i n y l greening subcellular photodynamic action, 123 group, description, 304 Dark m o n o v i n y l / l i g h t d i v i n y l g r o u p , d e s c r i p t i o n , 304

greening

Dark monovinyl/light d i v i n y l greening group,description, 304 Degradation, nonphotoactive natural pesticides, 171 cis-Dehydromatricaria ester, o v i c i d a l a c t i v i t y , 12 Dehydrosafynol, structure, 285 Delayed developmental periods of insects antifeedant, 148-149 delayed adult emergence, I47,l48t retardation of growth, 145,147

E

Ecological v a r i a t i o n to phototoxins geographic l o c a t i o n , 214 shade, 213 Enzymatic generation of s i n g l e t oxygen biosynthetic enzymes, 69 lactoperoxidase, 68 microbicidal enzymes, 66,68,70f plant enzymes, 68-69,70f techniques for study, 66

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

INDEX

333

Eosin, photodynamic effect on pea l e a f tissue, 9 Error-prone repair, description, 193 Erythrosin B a d u l t i c i d e tests against house f l i e s , I6l,l62t cross resistance of house f l i e s , 8 effect of l i g h t on culex larvae, 6 f i r e ant c o n t r o l , 163,165 l a r v i c i d e tests against house f l i e s , 158-159,I60t mosquito c o n t r o l , 162-163 photodynamic effect on house f l y , 7-8 t o x i c i t y to g a s t r o i n t e s t i n a l nematodes, 9 Escherichia c o l i (3-galactosidase a c t i v i t y , 198-199,200 DNA repair systems, 193-19 reasons for use i n studying phototoxins, 192 s t r a i n s , 195t Escherichia c o l i s t r a i n s , fluence-response curves, 195,196-197f,198 Excision repair, description, 193 Excitable c e l l s , photodynamic modification, 109-120

F Face f l y , t o x i c o l o g i c a l e f f e c t s of erythrosin B and rose bengal, 7 F a l c a r i n o l , structure, 285 F a t t y a c i d s v s . p r o t e i n as phototoxins, 201,202f

target

Fungal pathogens of Continued

citrus—

resistance of above-ground vs. root t i s s u e , 290 s u s c e p t i b i l i t y , 287 Furanocoumarins application as defense chemicals, 221 biochemical e f f e c t s , 220 description, 217,282 light-dependent actions, 219 light-independp^t actions, 21ft,226-227 medicinal uses, 221-222 metabolic transformations i n b i r d s , 222,225 insects, 225 mammals, 222 nonspecific

phototoxicity of angular vs. l i n e a r , 10 rate of metabolism, 226 role as plant defensive agents, 282-283 role i n plant t i s s u e s , 282 role of oxygen i n action, 219-220 s t r u c t u r e - a c t i v i t y relationship to photosensitizing a c t i v i t y , 220 structures, 217,2l8f,219 t o x i c o l o g i c a l e f f e c t s , 220 use as antifeedants, 221 phytoalexins, 221 Furans, use as nonspecific s i n g l e t oxygen traps, 29

of

Fecundity, d e f i n i t i o n , 149 F i r e ant control e f f i c i e n c y of b a i t s , I 6 3 , l 6 4 t , l 6 5 f i e l d t e s t s , 163-165 F l u i d mosaic model, structure of b i o l o g i c a l membranes, 40,42f Fluoranthene, phototoxicity, 198 Fluorescein derivatives, e f f e c t on developmental stages of house fly, 6 Fluorescent dyes, mosquito c o n t r o l , 162-163 Formaraide, s i n g l e t oxygen l i f e t i m e , 49,50f,51 Fungal c e l l w a l l , e f f e c t on cercosporin resistance, 276-277 Fungal pathogens of c i t r u s i n h i b i t o r y e f f e c t of coumar-ins and furanocoumarins, 290,291t crude l e a f extracts, 287,289t,290 l i s t , 287,288t

G Giant axons function and behavior, 111 photodynamic modification, 111-112 G l y c e o l l i n , structure, 286 Greening groups of higher plants, c l a s s i f i c a t i o n , 301,304 Greening patterns i n higher plants, molecular o r i g i n , 304-305 Greening process, use i n herbicide development, 296-297

H Halogenated fluorescein derivatives efficacy of photodynamic action against house f l y , 3 structure, 2,4

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

334

LIGHT-ACTIVATED PESTICIDES

Halogenated fluorescein s e n s i t i z e r s r e l a t i v e potency, 44,45f s e n s i t i z i n g e f f i c a c y , 44,46 Harmane a l k a l o i d s , structure and phototoxicity, 233 Hematoporphyrin-sensitized generation of s i n g l e t oxygen, photochemical and photobiological e f f e c t s of oxygen molecule, 64,65f H i g h - s e n s i t i v i t y luminescence spectrometer diagram, 59,60f s e n s i t i v i t y , 59 use i n s i n g l e t oxygen i d e n t i f i c a t i o n , 58-72 Higher plants, greening group c l a s s i f i c a t i o n , 301,304 H i s t i d i n e , 101 House f l y cross resistance, 8 photodynamic effect of rose bengal and erythrosin B, 7-8 House f l y control a d u l t i c i d e t e s t s , 160-162 l a r v i c i d e t e s t s , 157-160 House f l y larvae, enhancement of phototoxicity erythrosin B, 9 Hydroxylactam, diagnostic trap f o r s i n g l e t oxygen, 30 Hypericin chemistry, 265-266 d i s t r i b u t i o n , 266 effect on l i g h t s e n s i t i v i t y i n animals, 13 photodynamic action, 265-269 phototoxic action, 267-268,269t promotion of type I I photodynamic reactions,

267

s i n g T e t m o l e c u l a r oxygen

production, 267 structure, 13,16,265-266 Hypericism, d e f i n i t i o n , 265 I Illumination i n the presence of photosensitizers, effect on biochemistry of molecules, 98-106 Inactivation gating k i n e t i c s , 1l6,117f process, 114,116 Infrared luminescence measurement apparatus, 82-85 quantum y i e l d measurements, 84-88 s i n g l e t oxygen detection, 32-33 I n h i b i t o r s , characterization of s i n g l e t oxygen, 31 Insects physiological effects of photodynamic action, 122-130 subcellular photodynamic action, 123-124

I n t e r f a c i a l region of membrane, description, 41 Intermediate photosensitizers, examples, 234-235 K K h e l l i n , structure and phototoxicity, 233 Khellin-thymine adduct, structure and phototoxicity, 233 L Lactoperoxidase , y , Laser herbicides, e f f e c t on chlorophyll biosynthesis, 13-15 Lifetimes, s i n g l e t oxygen, 49,50f,51 Light energy use f o r defensive purposes, 207 use i n t o x i c o l o g i c a l reactions, 1 Light-activated pesticides, photodynamic modification, 119 Light-dependent t o x i c i t y mechanisms photodynamic action, 135-140 photodynamic damage, 135 photosensitizers, 135 Light-independent t o x i c i t y mechanism biochemical changes, 141 c h a r a c t e r i s t i c s , 140 xanthene dyes, 140 Light-induced f i r i n g of nerve c e l l s a c t i v a t i o n , 118 perturbations i n behavior, 118 r e v e r s i b i l i t y , 118 L i p i d s , photodynamic e f f e c t s , 100-101 Liposomes, description, 41 Lipoxygenase, s i n g l e t oxygen generation, 69 Lobster giant axons, photodynamic modification, 113-116 Luminescence, s i n g l e t oxygen detection, 32,34f Luminescence detection, s i n g l e t oxygen, 8 0 , 8 l f Lysine effect of phototoxins, 126 photodynamic e f f e c t s , 102

M Mechanistic studies on photodynamic pesticides cercosporin, 33-34 t e r t h i e n y l , 35-36

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

INDEX

335

Membrane environment, features, 40-41 Membrane model systems, micelles and liposomes, 4l,42f Membrane photomodification e f f e c t of membrane f l u i d i t y , 53-54 effect of temperature, 53-54 reasons for study, 39 Merocyanine 540, use as s e n s i t i z e r , 43-44 5-Methoxypsoralen, structure, 283 8-Methoxypsoralen, structure, 283 Methylene blue, phototoxicity, 3 Methylene blue sensitized generation of s i n g l e t oxygen, photochemica* and photobiological effects of oxygen molecule, 6l,64,65f Microbial enzymes, s i n g l e t oxygen generation, 68,70f Mismatch repair, description Modification of axon function photochemical mechanisms, 119 Molecular oxygen i n d i r e c t methods of production, 76 lowest excited state, 76 single-state sources, 78 solvent i n t e r a c t i o n , 6 l - 6 2 f , 6 3 f t r i p l e t - s t a t e sources, 78-79 Monoacetylenes, UV absorption spectra, 249,250f 1

Neuromuscular transmission, vertebrates, 110-111

process

in

Nonphotoactive natural pesticides, degradation, 171 Nucleic acids, photodynamic e f f e c t s , 104

Operon fusion s t r a i n s , studies of gene regulation, 198 Ovicidal a c t i v i t y , 12 Oxidative stress repair system, description, 194

Pesticide technology, state of the art i n 1950s, 2 Phaseollin, structure, 286 Phenylalanine, photodynamic e f f e c t s , 102 Monovinylprotochlorophyllide accumulatiorip i n h i b i t o r s , 318,323,324-326t made of toxic action, 10,12 Morphological abnormalities o v i c i d a l a c t i v i t y , 12 e f f e c t of phototoxicity to insects, 256 molting hormones, I 4 5 , l 4 6 f structure, 10-11,285 sensitizers, I44t,l45 Phloxin B, f i r e ant control, 163,165 examples, I 4 2 , l 4 3 f , l 4 4 Photoactivated t o x i c i t y of influencing factors, 142 cercosporin, mechanism, 272-273 Mosquito control, f i e l d t e s t s , 162-163 Photoactive compounds, naturally Muscle membrane, photodynamic occurring, structures, 168,170 action, 111 Photoactive plant components, Myeloperoxidase studies, 9 antimicrobial c h a r a c t e r i s t i c s , 66,68 Photoactive substances on insects mechanism of action, 68 development, 128-129 s i n g l e t oxygen generation, 68,70f reproduction, 129-130 Photoactivity of o - t e r t h i e n y l in fish N mechanism, 181 survival p r o f i l e , I 8 l , l 8 4 f testing procedures, 181 Natural phototoxins, i n s e c t i c i d a l i n tadpoles properties, 207,208t survival p r o f i l e s , I 8 l , l 8 2 f Naturally occurring and synthetic testing procedure, 180-181 acetylenes, phototoxicity against i n water f l e a s , s u r v i v a l microoganisms, 246,247t p r o f i l e s , I83,l84f Naturally occurring photosensitizers, Photobiological actions of fungicidal a c t i v i t y , 231-237 furanocoumarins, DNA Nerve c e l l experiments, photoalkylation, 219 observations, 116,118-119 Photochemical systems, Nerve-muscle preparations, characterization of s i n g l e t photodynamic studies, 110-111 oxygen, 31-32 Nervous systems Photodecomposition, naturally elemental processes, 110 occurring biocides, 168-173 function, 109 nen

l h

D t a t r i v n e

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

336

LIGHT-ACTIVATED PESTICIDES

Photodynamic action biochemistry, 46-47 d e f i n i t i o n , 2,22 hypericin, 265-269 primary target s i t e s , 136-140 type I and type I I mechanisms, 22-36 use against an i n s e c t , 2 Photodynamic action on biochemica components dye binding, 136 photosensitizer binding s i t e , 136-137 glutathion l e v e l s , 137 photoalteration, 136-137 Photodynamic action on b i o l o g i c a l membranes c e l l u l a r damage, 138 h i s t o l o g i c a l and physiological damage, 138 photochemical damage, 138-13 volumetric changes i n hemolymp crop contents, 138 Photodynamic action on enzyme systems, i n a c t i v a t i o n , 139 Photodynamic dyes i n insects, dark reaction, 3 Photodynamic e f f e c t s alcohols and carbohydrates, 100 amino acids, 101-102 biomolecules, 104-105 i n solution vs. i n c e l l s and organelles, 104-105 l i p i d s , 100-101 proteins, 102-103 purines, 103-104 pyrimidines, 103-104 Photodynamic herbicide system development of the concept, 296-297 herbicide choice, 297,298-299f p r i n c i p l e s and guidelines, 295-296 Photodynamic modification giant axons, 111 l i g h t - a c t i v a t e d species, 119 Photodynamic modification of lobster giant axons block of sodium channels, 113-114 perturbation of potassium channels and leakage, 116 sodium channel gating, 1l4,115f,1l6,117f Photodynamic reactions, p r i n c i p l e mechanisms, 99 Photodynamic studies muscle membranes, 111 nerve-muscle preparations, 111 Photodynamic t o x i c i t y , effect of l i g h t i n t e n s i t y , 5-6 Photodynamically active compounds, mutagenicity, 124 125 Photoexcitation of s e n s i t i z e r , perturbation e f f e c t , 76-77 1

Photoinsecticidal a c t i v i t y of a-terthienyl procedure t e s t i n g , 178 s u r v i v a l , p r o f i l e s , 178,179f,l80 Photooxidation reactions, mechanisms involving s i n g l e t oxygen, 59,61 Photooxidative dyes l a r v i c i d e tests against flies, 157-160

house

mammalian t o x i c i t y , 157t rapid degradation, 157 Photooxidative dyes as i n s e c t i c i d e s , f i e l d development, 156-165 Photooxygenation mechanisms, 22-26 methods for mechanism determination, 27-29 Photoreactivation, d e s c r i p t i o n , 193

studying, 256 Photosensitized oxidations, applications, 78 Photosensitized oxidations i n complex systems, establishing the mechanism, 28-29 Photosensitizers e f f e c t s on insect hemocytes, 125-126 whole c e l l s , 125 evolution as a defense mechanism, 255 intermediate, 234-235,237f mechanisms o f a c t i o n, 231-232 type I , 232-234,237f type I I , 236,237f Photosensitizers f o r b i o l o g i c a l systems, e f f e c t i v e compounds, 99 Photostability of dyes, influencing factors, 171-172 Phototoxic chemicals, effects on insects, 256 Phototoxicity, e f f i c a c y as a defense against insects, 209 Phototoxicity assays microorganisms, 247,248t standard bioassays, 247-248 Phototoxicity of a - t e r t h i e n y l , b i o l o g i c a l mechanism, 183 Phototoxin behavioral resistance, 209,210t evaluation of mutagenesis, 195 protection of c e l l by cloned carotenoid genes, 201,203 Phototoxins biochemical resistance, 212,213t characterization of effects, 194-195

ecological f a c t o r s , 213-214 fatty acid auxotroph with Escherichia c o l i , 199,201 physical resistance, 211

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

INDEX

337

Physical resistance to phototoxins highly r e f l e c t i v e surfaces, 211-212 melanin concentrations, 211 Physiologica effects of photodynamic action adverse e f f e c t s , 128-130 c e l l u l a r l e v e l , 125-126 subcellular l e v e l , 123-125 systems l e v e l , 126-128 P i s a t i n , structure, 286 Plant defensive agents, photosensitizers, 282-286 Plant enzymes, singDet oxygen generation, 68-69,70f Polyacetylene effect as pesticide, 12 mechanisms of action, 249,251 structure, 249 Polyacetylenephenylheptatriyne structure and phototoxicity, 234-23 Polyacetylene photoactivity, structure and function r e l a t i o n s h i p s , 241-251 Polyacetylenes aerobic conditions, 248 description, 241,283 e f f e c t on membrane permeability, 173 light-independent b i o l o g i c a l e f f e c t s , 242 nonphotoactive, 242,244t phototoxicity, 173 phototoxicity against microorganisms, 248t,249 phototoxicity against microorganisms under anaerobic conditions, 251t phototoxicity to insects, 255-256 role as photosensitizers, 283 plant defensive agents, 283,284t,285 structures, 241 ,243f Polyunsaturated fatty acids, diagnostic trap f o r s i n g l e t oxygen, 30 Porphyrins and S values, 92,95t s e n s i t i z i n g e f f i c a c y , 44,46 Potassium channels i n excitable c e l l s photodynamic perturbation, 116 types, 116 Proteins, photodynamic e f f e c t s , 102-103 Pterocarpans description, 286 role as plant defensive agents, 286 Pulsed e x c i t a t i o n , apparatus, 84,85f Purines, photodynamic e f f e c t s , 103-104 Pyrethrins, photodegradation, 171 Pyrimidines, photodynamic e f f e c t s , 103-104 1

Q Quantum y i e l d measurements c a l i b r a t i o n of s i n g l e t oxygen luminescence s i g n a l , 84,86,87f concentration determination, 86,88 Quantum y i e l d s measurement f o r s i n g l e t oxygen, 77 s i n g l e t oxygen, 47,48f,49 Quenching, of s e n s i t i z e r excited states, 28 R

Recombinational repair, description, 193 -276 fungal c e l l w a l l , 276-277 influencing factors, 275 mechanism, 274-275 Reverse Diels-Alder reaction, characterization of s i n g l e t oxygen, 31-32 Rose bengal effect of t o x i c i t y on mosquitoes, 6 effect on locomotary a c t i v i t y of house f l y , 139t,l40 photodynamic effect on house f l y , 7-8 Rose bengal derivatives, values, 92,94t Rotenone, photodegradation, 171 S

S values porphyrins, 92,95t s e n s i t i z e r s i n solvents, 88,89t,91t S e l e c t i v i t y of a-terthienyl as a phototoxic i n s e c t i c i d e problems with data i n t e r p r e t a t i o n , 186 rate of depuration, I87,l88f r o l e of k i n e t i c s , 186 s u r v i v a l p r o f i l e s , I89f S e n s i t i v i t y of ar-terthienyl, l a s t i n s t a r larvae, 257t Sensitizer excited states, quenching, 28 Sensitizer-membrane interactions e f f e c t on s i n g l e t oxygen generation by s e n s i t i z e r s , 41,43 s e n s i t i z e r associated with membrane, 44,45f,46 s e n s i t i z e r bound to membrane, 43-44 s e n s i t i z e r external to membrane, 43

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

338

LIGHT-ACTIVATED PESTICIDES

Sensitizers degree of halogenation vs. quantum y i e l d s vs. effectiveness, 49 photoexcitation, 76-77 S values, 88,89t,91t Singlet oxygen characterization techniques, 29-36 d i f f u s i o n across membranes, 51,52f,53 e f f e c t on photoactivity of xanthene dyes, 172 electronic energy transfer generation, 59,61 i d e n t i f i c a t i o n i n reactions, 58-72 i d e n t i f i c a t i o n problems with secondary evidence, 58 l i f e t i m e s , 49,50f,51 measurement of quantum y i e l d s , 77-78 photosensitized emission dyes, 6l,64,65f photosensitized generation, quantum y i e l d s , 47,48f,49 quenching, 26,28 rate of formation, 79 types of luminescence, 26 y i e l d measurements, 79-82 Singlet oxygen generation s e n s i t i z e r associated with membrane, 44,45f,46 s e n s i t i z e r external to membrane, 43 Singlet oxygen generation i n membranes, c h a r a c t e r i s t i c features, 39 Singlet oxygen i d e n t i f i c a t i o n , h i g h - s e n s i t i v i t y luminescence spectrometer, 59,60f Singlet oxygen i n membranes, c h a r a c t e r i s t i c s , 46-54 Singlet oxygen modification of membranes, biochemistry, 46-47 Singlet state, sources, 78 Sodium channels action, 113 blockage, 113-114 disturbance of i n a c t i v a t i o n , 1l4,115f subpopulations, 1l4,115f Structure function r e l a t i o n s h i p s , polyacetylene photoactivity, 245-219 Subcellular photodynamic action, physiological e f f e c t s , 123-124 Sunlight effect on chemical l e v e l , 207 e f f e c t on metabolic rates, 207,208t Synerid a d u l t i c i d e tests against house f l i e s , 160-161,I62t l a r v i c i d e tests against house f l i e s , 158 Synerid 100, l a r v i c i d e tests against house f l i e s , 158-159

Synthetic dyes, t o x i c i t y mechanisms, 134-152 Systems photodynamic action physiological e f f e c t on body f l u i d s , 127 physiological effect on components of body f l u i d s , 126-127 physiological e f f e c t on nervous system, 127-128

T a-Terthienyl biologica , effect of l i g h t on t o x i c i t y , 12 environmental safety, 259 l a r v i c i d a l e f f i c a c y vs. l i g h t and time, 258,259t mode of toxic action, 10-11 nematicidal a c t i v i t y , 10 photodynamic e f f e c t s , 35 photoinsecticidal a c t i v i t y , 178-180 phototoxic e f f e c t s , 256 phototoxicity in f i s h , I 8 l , l 8 4 f in tadpoles, 180-181,l82f in water f l e a , I83,l84f s e l e c t i v i t y as a phototoxic i n s e c t i c i d e , 186-187,I88f s e n s i t i v i t y , 257t s i n g l e t oxygen detection, 35-36 synthesis, 258 structure, 10-11,176,177f,241,243f,285 t o x i c i t y , 176 t o x i c i t y to nontarget organisms, 259,260t ff-Terthienyl analogues and derivatives e f f e c t of l i g h t , 26l,262t mechanisms of a c t i o n , 261 structures, 260 t o x i c i t y , 260-261 Tetracyclines, phototoxicity, 64,66,67f Tetrapyrrole accumulation dependence of photodynamic damage, 306-307 dependence of photodynamic damage on the chemical nature, 307 Tetrapyrroles, stimulation by herbicides, 14 Thermal generation of s i n g l e t oxygen, d i s s o c i a t i n g endoperoxide, 69,71f Thermal lensing, description, 82 Thiarubrines structure and phototoxicity, 236

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

339

INDEX

Thiophenes photodegradation, 173 phototoxicity to insects, 255-256 sources, 256 Time-resolved conductivity measurements, s i n g l e t oxygen detection, 33,34f Toxicological e f f e c t , early history of l i g h t enchancement, 3 Transforming deoxyribonucleic a c i d , effects of phototoxins, e f f i c a c y for i n a c t i v a t i o n of transforming a c t i v i t y , 203 Transient absorption spectroscopy schematic of system, 33,34f s i n g l e t oxygen detection, 33 4,5 ,8-Trimethylpsoralen. metabolites, 222,224f T r i p l e t state, sources, 78-7 T r i t i a t e d phototoxins e f f e c t of t o p i c a l a p p l i c a t i o n , 257 production, 257 Tryptophan, photodynamic e f f e c t s , 101-102 Type I mechanism of photosensitized oxidation electron promotion i n excited state, 23,24f factors governing competition with type I I mechanism, 23,27 mechanism, 23f oxidation of aromatic o l e f i n s , 23,25 process, 23 Type I photosensitizers, description, Type I reactions, mechanism, 99,231 Type I I mechanism of photosensitized oxidation additions to o l e f i n s with a l l y l i c hydrogens, 25 deactivation of s i n g l e t oxygen, 25 dienes, aromatics, and heterocyc]es, 25 electron-rich o l e f i n reactions, 26 electron-rich phenol reactions, 25 factor governing competition with type I mechanism, 23,27 mechanism, 23f oxidation of s u l f i d e s , 26 process, 23 s i n g l e t molecular oxygen production, 25 s i n g l e t oxygen quenching, 26 Type I I photosensitizers, examples, 236 Type I I reactions, mechanism, 99,231-23i Tyrosine, photodynamic e f f e c t s , 102 1

V Values porphyrins, 92,95t rose bengal derivatives, 92,94t s e n s i t i z e r s i n solvents, 88,90t,91t xanthene dyes, 92,93t V i t a l enzyme systems, effect by photodynamic action, 139-140 Vitamin C, quenching of s i n g l e t oxygen, 64,65f-66f Voltage clamp analysis of membrane channel function sodium channels, 113 technical background, 112-113

Water-soluble porphyrins and metallic derivatives, values, 92,95t Wyerone, structure, 285

X

:

Xanthene dyes dependence on phosphorescence quantum y i e l d , 5 effect on insect reproduction, 129-130 nervous system, 127-128 light-independent t o x i c i t y , 140 photodegradation, 172 r e s t i v e t o x i c i t i e s to mosquito larvae, 8 structures, 168,170 t o x i c i t y , 3,172 values, 9 2 , 9 3 t Xanthotoxin i n vivo and i n v i t r o metabolism, 222,223f phototoxicity, 9-11 Y Yield measurement of s i n g l e t oxygen, chemical traps, 79-80

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.