Light-Activated Pesticides (ACS Symposium Series 339)

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

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.


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


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


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


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.


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


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


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


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.


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


LIGHT-ACTIVATED PESTICIDES

A

B

H Br I

H H H

DYE Fluorescein Eosin Erythrosin B

Structure I

Methylene Blue

Structure I I


1.

HEITZ


( 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.


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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


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HEITZ


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


10


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


1.

HEITZ

11


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


12


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 .


1.

HEITZ


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


14


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


1.

HEITZ


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.



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


1. HEITZ


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.


18 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. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49.


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.


1. HEITZ


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.


19

20


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.


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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


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


2.

FOOTE

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 •

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 ) .



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)


2.

FOOTE

27


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


28


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


2.

FOOTE

29


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


30


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.


31


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


32


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 1.27îm 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



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



(

N Computer

\

Digitizer Signal Avg.

I-Amplifiers

\/ Filters

| — Photodiode

Laser

-d

—Cell

Fig. 3. Singlet Oxygen Detection

Computer v

^

iÂmplifiers 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


2.

FOOTE

35


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


36


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

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


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


40 The


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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Photomodification and Singlet Oxygen Generation

41

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


42


Intrinsic Anion Transport ^Proteins Protein |r

Ankyrin

, 6 7 ). 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


52


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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53

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



54

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 . ]


3.

VALENZENO


55

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.


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.


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.


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.


57

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


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 (Âg) 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.



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

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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


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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 (-Âg) 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

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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


1.20

1.30

1.40

1.50

1.60

1.40 1.50

1.60

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.)


4. KHAN


63

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1.60

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


1 8

64


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



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1200

800

1600

800

1200

1600

1200

800

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

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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 ) .



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



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 ) .


68


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


4. KHAN

69


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 (Âg), 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



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) .


4.


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 ) .


72


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

Âg)

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 ) .

Literature Cited 1. Singlet Molecular Oxygen. Benchmark Papers in Organic Chemistry: Schaap, A. P., Ed.; Dowden, Hutchinson and Ross: Stroudsburg, PA, 1976; Vol. 5. 2. Singlet Q ; Frimer, A. A., Ed.; CRC Press, Inc.: Boca Raton, FL, 1985; Vol. 1-4. 3. Khan, A. U.; Kasha, M. Proc. Natl. Acad. Sci. USA 1979, 76, 6047-49. 4. Khan, A. U. J . Am. Chem. Soc. 1981, 103, 6516-17. 5. Krasnovsky, A. A., Jr. Biofisika 1976, 21, 748-49. 6. Byteva, I. M.; Guvinovitch, G. P. ZPrikl. Spektr. 1978, 29, 154. 7. Khan, A. U . ; Kasha, M. J. Chem. Phys. 1963, 39, 2105-6. 8. Khan, A. U.; Kasha, M. J. Am. Chem. Soc. 1970, 92, 3293-300. 9. Arnold, S. J.; Ogryzlo, E. A.; Witzke, H. J. chem. Phys. 1964, 40, 1769-70. 10. Wasserman, H. H . ; Larsen, D. L. JCS Chem. Comm. 1972, 253-54. 11. Turro, N. J.; Chow M. -F.; Rigaudy, J . J . Am. Chem. Soc. 1981, 103, 7218-24. 12. Krasnovsky, A. A., Jr. Chem. Phys. Lett. 1981, 81, 443-45. 13. Fritzsche, M. C. R. Acad. Sci. (Paris) 1867, 64, 1035-37. 14. Raab, O. Z. Biol. 1900, 39, 524-46. 15. Kautsky, H . ; de Bruijn, H. Naturwissenschaften 1931, 19, 1043. 2


4. KHAN


73

16. Gaffron, H. Biochem. Z. 1936, 287, 130-39. 17. Schenck, G. O. Naturwissenschaften 1948, 35, 28-29. 18. Khan, A. U. In Singlet O ; Frimer, A. A., Ed.; CRC Press, Inc.: Boca Raton, FL, 1985; Vol. 1, Chapter 3. 19. Foote, C. S.; Wexler, S. J . Am. Chem. Soc. 1964, 86, 3879-80. 20. Corey, E. J.; Taylor, W. C. J. Am. Chem. Soc. 1964, 86, 388182. 21. McKeown, E.; Waters, W. A. J . Chem. Soc. (B) 1966, 1040-46. 22. Childs, W. H. J.; Mecke, R. Z. Phys. 1931, 68, 344-61. 23. Badger, R. M.: Wright, A. C.; Whitlock, R. F. J . Chem. Phys. 1965, 43, 4345-50. 24. Kawaoka, K.; Khan, A. U.: Kearns, D. R. J. Chem. Phys. 1967, 46, 1842-53. 25. Kawaoka, K.; Khan, A. U . : Kearns, D. R. J . Chem. Phys. 1967, 47, 1883-84. 26. Snelling, D. R. Chem. Phys. Lett. 1968, 2, 346-48. 27. Kearns, D. R.; Khan, A. U.; Duncan, C. K.; Maki, A. H. J.Am.Chem.Son. 1969 28. Wasserman, E . ; Kuck J.Am.Chem.Soc. 1969 29. Khan, A. U. Chem.Phys.Lett. 1980, 72, 112-14. 30. Chou, P.; Khan, A. U. Chem.Phys.Lett. 1984, 103, 281-84. 31. Gollnick, K. Adv.Photochem. 1968, 6, 1-122. 32. Spikes, J . D.; Livingston, R. Adv.Radiation Biol. 1969, 3, 29121. 33. Monroe, B. In Singlet O ; Frimer, A. A. ED.; CRC Press Inc.: Boca Raton, FL, 1985; Vol.1 Chapter 5. 34. Khan, A. U.; Kasha, M. Nature 1964, 204, 241-43. 35. Dougherty, T. J.; Kaufman, J . E.; Goldfarb, A , ; Weishaupt, K. R.; Boyle,D.; Mittleman, A. Cancer Res. 1978, 38, 2628-35. 36. Krasnovsky, A. A. J r . Photochem. Photobiol. 1979, 29, 29-36. 37. Selkirk, J . K. In Modifiers of Chemical Carcinogenesis: An Approach to the Biochemical Mechanism and Cancer Prevention; Slaga, T. J . ED.; Raven Press: New York. 1980; Chapter 1. 38. Khan, A. U.; Kasha, M. Ann. N. Y. Acad. Sci. 1970, 171, 24-32. 39. Chou, P.; Khan, A. U. Biochem.Biophys.Res.Commun. 1983, 115. 932-37. 40. Szent-Györgyi, A. Studies on Biological Oxidation. Barth: Leipzig, Germany, 1937. 41. Lind Bicentenary Symposium: Stewart, C. P. ED.; Edinburgh, Scotland, 1953. 42. Vitamin C: Burns, J . J . ED.; Ann. N. Y. Acad. Sci. 1961, Vol.92, Art.1. 43. Pauling, L. How to Live Longer and Feel Better: W.H.Freeman, New York, 1986. 44. Gale, E. F . ; Cundliffe, E . ; Reynolds, P . E . ; Richmond, M. H.; Waring M. J. The Molecular Basis of Antibiotic Action: Wiley, New York, 1981; pp 448-53. 45. Cullen, S. I.; Catalano, P. M.; Helfman, R. J . Arch. Dermatol. 1966, 93, 77. 46. Schorr, W. F.; Monash, S. Arch Dermatol. 1963, 88, 134-38. 47. Frost, P.; Weinstein, G. D.; Gomez, E.C. JAMA 1971, 216, 32629. 48. Zuehlke, R. L. Arch Dermatol. 1973, 108, 837-38. 49. Frank, S. B.; Cohen, J . H . ; Minkim, W. Arch. Dermatol 1971, 103 520-21. 50. Blank, H.; Cullen, S. I.; Catalano, P. M. Arch. Dermatol. 1968, 97, 1-2. 51. Weibe, J . A. : Moore, D. E. J . Pharm. Sci. 1977, 66 186-89. 2

2


74


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.


4. KHAN


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


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



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( î)

~

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 -ETÊA). 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


78


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


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

M0

g

2

> product


(13)

80


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


RODGERS

1200


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.


82


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


RODGERS


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). 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).


9.

PIMPRIKAR AND COIGN

Multiple Mechanisms of Dye-Induced Toxicity

139

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


140


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 ) .


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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 ) ,


142


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



Multiple Mechanisms of Dye-induced Toxicity

143

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.


144


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


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.

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

148


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 .


9.

PIMPRIKAR AND COIGN


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


150


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).




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 .


151

152


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

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

Bakker, J.; Gommers, F.J.; Nieuwenhuis, I.; Wynberg, H. J . Biol. Chem. 1979, 254, 1841-1844. Lochmann, E.R.; Micheler, A. In Physico-chemical Properties of Nucleic Acids; Ducheswe, J. Ed.; Academic Press, New York, 1973;1, Chapter 8. Spikes, J . D . ; Livingston, R. Adv. Radiat. Biol. 1969, 3, 29-121. Spikes, J.D. In The Science of Photobiology; Smith, K.C., Ed.; Plenum, New York, 1977; 87-112. Foot, C.S. In Free Radicals in Biology; Pryor, W.A., Ed.; Academic, New York, 1976; 85-133. Robinson, J.R.; Beatson, E.P. Pest. Biochem. Physiol. 1985, 24, 375-383. Amagasa, J. Photochem. Photobiol. 1981, 33, 947-955. Bezman, S.A.; Burtis, P.Α.; Izud, T . P . J . ; Thayer, M.A. Photochem. Photobiol. 1978, 28, 325-329. Miyushi, N.; Tomita, G. Photochem. Photobiol. 1979, 29, 527-530. Usui, Y. In Singlet oxygen; Rawby, B.; Rabek, J.F., Eds.; Wiley, Chichester, England, 1978; 203-210. Nieumint, A.W.M.; Aubry, J . M . ; Arwert, C.; Kortbeck, H . ; Herzberg, S.; Joenje, H. Free Radical Res. Communications, 1985, 1, 9. Glazer, A.N. Proc. Natl. Acad. Sci. U.S. 1970, 65, 1057-1063. Straight, R.C.; Spikes, J.D. In Singlet oxygen; Frimer, A . A . , Ed.; CRC, Boca Raton, Florida; 1985; 4, 91-143. 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; 441-459. Lamola, A.A.; Yamane, T . ; Trozzolo, A.M. Science, 1973, 179, 1131-1133. Ito, T. Photochem. Photobiol. 1978, 28, 493-508.



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.


153

Song, P.S.; Tapley, K.J., J r . Photochem. Photobiol. 1979, 29, 1177-1197. Bellin, J . S . ; Grossman, L . C . Photochem. Photobiol. 1965, 4, 45-53. Philogene, B.J.R.; Arnason, J . T . ; Duval, F. Can. Entomol. 1985, 117, 1153-1157. Arnason, J . T . ; Fortier, G . ; Champagne, D.; Philogene, B.J.R. Rev. Can. Biol. Exp. 1983, 42, 205-208. Pathak, M . A . ; Kramer, D.M.; Fitzpatrik, T.B. In Sunlight and Man; Pathak, M.A.; Harber, L.C.; Seiji, M.; Kukita,A.; Eds.; University of Tokyo Press, Tokyo, 1974; pp.335. Wages, J . Ph.D. Dissertation Mississippi State University, Mississippi State, 1985. Miller, A . C . ; Henderson, B.W. Radial. Res. 1986, 107, 83-94. Yoho, T.P. Ph.D. Dissertation, West Virginia University, Morgantown, 1972. Barbieri, A. Riv. Weaver, J.E.; Butler 5, 840-844. Yu, B.P.; Masuro, E.J.; Bertrand, H.A. Biochem. 1974, 13, 5083-5087. Takahara, J.; Yunoki, S.: Yamauchi, J.; Yakushiji, W.; Hashimoto, K; Ofuji, T. Life Science, 1981, 29, 1229-1233. Duncan, C . J . ; Bowler, J . J. Cell Physiol. 1970, 79, 259-271. MacRae, W.D.; Irwin, D.A.; Bisalputra, T . ; Towers, G.H.N. Photochem. Photobiophys. 1980, 1, 309-318. Downum, K.R.; Hancock, R.E.W.; Towers, G.H.N. Photochem. Photobiol. 1982, 36, 517-523. Freeman, P . J . ; Giese, A.C. J. Cellular Comp. Physiol. 1952, 39, 301-322. Pooler, J . P . ; Valenzeno, D.P. Biochem. Biophys. Acta, 1979, 555, 307-315. Allison, A.C.; Magnus, I . Α . ; Young, M.R. Nature, 1966, 209, 874-878. Cande, W.Z.; McDonald, K.; Meeusen, R.L. J. Cell Biol. 1981, 88, 618-629. Haga, J . Y . ; Spikes, J.D. In Organic Biological and Medicinal Chemistry; Galo, U . ; Santamaria, L., Eds.; American Elsevier Publishing Co., New York, 1972; 3, 464-479. Daub, M.E.; Briggs, S.P. Plant Physiol. 1983, 71, 763-766. Wagner, S.; Taylor, W.D.; Keith, Α.; Snipes, W. Photochem. Photobiol. 1980, 32, 771-779. Ito, T. Photochem. Photobiol. 1980, 31, 565-570. Tudball, N.; Thomas, P. Biochem. J. 1971, 123, 421-426. Brand, K . ; Tsolas, O.; Horecker, B.L. Arch. Biochem. Biophys. 1969, 130, 521-529. Rosenthal, I. Photochem. Photobiol. 1976, 24, 641-645. Rahimtula, A.D.; Hawco, F.J.; O'Brien, P . J . Photochem. Photobiol. 1978, 28, 811-815. Carraro, C.; Pathak, M.A.; Bissett, D.L. Photochem. Photobiol. 1986, 43, 14S. Tsai, C.S.; Godin, J.: Wand, A . J . Biochem. J . 1985, 225, 203-208.


154 46. 47. 48. 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. 76. 77.

LIGHT-ACTIVATED PESTICIDES Garcia, F.J.; Yamamoto, E.; Abramowski, Z . ; Downum, K.; Towers, G.H.N. Photochem. Photobiol. 1984, 39, 521-524. Knox, J . P . ; Dodge, A.D. Planta, 1985, 164, 22-29. Kaye, N.M.C.; Weitzman, P.D.J. FEBS Lett. 1976, 62, 334-337. F u . , N.; Yeh, S.; Chang, C.; Zhao, X.; Chang, L. Adv. Exp. Med. Biol. 1985, 193, 161-167. Gommers, F.J.; Bakker, J.; Smits, L. Nematologica, 1980, 26, 369-375. Callaham, M.F.; Lewis, L . A . ; Holloman, M.E.; Broome, J.R. Heitz, J.R. Comp. Biochem. Physiol. 1975, 51C, 123-128. Callaham, M.F.; Palmertree, C.O.; Broome, J . R . ; Heitz, J.R. Pest. Biochem. Physiol. 1977, 7, 21-27. Broome, J . R . ; Callaham, M.F.; Poe, W.E.; Heitz, J.R. Chem. Biol. Interact. 1976, 14, 203-206. Callaham, M.F.: Broome, J . R . ; Poe, W.E.; Heitz, J.R. Environ. Entomol. 1977, 6, 669-673. Blum, H.F. J. Invest Dermatol 1941 4 159-173 Broome, J . R . ; Callaham Heitz, J.R. Comp. Biochem Fondren, J.E., Jr.; and Heitz, J.R. Environ. Entomol. 1978, 7, 843-846. Fondren, J.E., Jr.; Heitz, J.R. Environ. Entomol. 1979, 8, 432-436. Creighton, C.S.; McFadden, T . L . ; and Schalk, J.M. J. Ga. Entomol. Soc. 1980, 15, 66-68. Carpenter, T . L . : Hetiz, J.R. Environ. Entomol. 1981, 10, 972-976. Respicio, N.C.; Heitz, J.R. Bull. Environ. Contam. Toxicol. 1981, 27, 274-281. 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. 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. Berenbaum, M. Science, 1978, 201, 532-534. Champagne, D . E . ; Arnason, J . T . ; Philogene, B.J.R.; Campbell, G.; Malachlan, D.G. Experientia, 1984, 40, 577-578. Yamamoto, E.; Wat, C.K.; MacRae, W.D.; Towers, G.H.N.; Chan, G.F.Q. FEBS Lett. 1979, 107, 134-136. Tauton, M.T.; Khan, S.M. Aust. J . Zool. 1978, 26, 139-146. Waldbauer, G.P. Adv. Insect. Physiol. 1968, 5, 229-288. Champagne, D . E . ; Arnason, J . T . ; Philogen, B.J.R.; Morand, R.; Lam, J. J. Chem. Ecol. 1986, 12, 835-858. Jordan, T.W.; Smith, J.N. Xenobiotica, 1981, 11, 1-7. David, J. Bull. Biol. France Belgique, 1963, 97, 515-530. Kolyer, J.M. J. Res. Lep. 1966, 5, 136-152. Barbosa, P.; and Peters, T.M. Entomol. Exp. Appl. 1970, 13, 293-299. Bridges, A.C.; Cocke, J.; Olson, J . K . ; Mayer, R.T. Mosquito News. 1977, 37, 227. Pimprikar, G.D.; Norment, B.R.; and Heitz, J.R. Environ. Entomol. 1979, 8, 856-859. Fairbrother, T . E . Ph.D. Dissertation, Mississippi State University, Mississippi State, 1978. Berenbaum, M.; Feeny, P. Science, 1981, 212, 927-929.


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


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


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


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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 .


158


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


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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


160


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


10.

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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


162


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


10.

LEMKE ET AL.


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. 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


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.

Cited

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.


166 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

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.


LEMKE ET AL. 32. 33. 34. 35.


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


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



MARCHANT

Photodecomposition of Naturally Occurring Biocides

IX

CH -CH»CH-(C«C) -(CH=CH) (CH ) 3

2

2

2

4

CH-CH

2

X

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

t



B = CI Br, B = CI

A=I, A =

A=I,

A=

B=H

Br, B B

A= H,

XI XH

xm

=H

JN

= H

XV

F i g u r e 2. S y n t h e t i c xanthene dyes. X I . Rose B e n g a l , X I I . P h l o x i n B, X I I I . E r y t h r o s i n B, XIV. E o s i n Y e l l o w , XV. Fluorescein.


11.

MARCHANT

Photodecomposition of Naturally Occurring Biocides

171

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 (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




266

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).


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



268

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


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



270

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.



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



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


20.

DAUB

Cercosporin's Role in Plant Disease

273

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


274


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 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


45-48

30,49-52

21.

DOWNUM AND NEMEC

Light-Activated Antimicrobial Chemicals

285

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


286


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


21.

DOWNUM AND NEMEC


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



[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



+

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


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



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.

292


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

Literature Cited 1.

2.

3.

4. 5. 6. 7. 8.

9. 10. 11. 12. 13. 14. 15.

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.


21.

DOWNUM AND NEMEC

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.


293

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.


294 47. 48. 49.

50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63.


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.



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


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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


22.

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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


298


I PROTOCHLOROPHYLL a. R = _ C H = C H ; R 2

2

=_CH ;R

3

3

(iDE)S = F.AI; D V 7 . F A I . E , P c h l

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

Pchl

l

= H ; D V , 7 _ C O O H I O _ C 0 M e Pchlide l

2

l

= A l k ; D V , 7 _ A l k . E , I O _ C O O H Pchlide

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 . )


22.

I.

299


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)

; R = F.AI; 2_MV, 7.FAI.E , 6 M e . P Mg Proto ( M V Mg Proto dieste

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

MgProto 6 M E )

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

Proto (DV

MgProto 7ester)

2

2

2

3

2

3

3

4

l

4

l

l

e. R = _ C H - C H ; R = _ C H ; R = H , 2 _ M V 7 _ C O O H , 6 M e . P , M g Proto(MV 2

2

3

3

f. R = _ C H _ C H ; R 2

2

3

3

2

2

h. R = _ C H _ C H ; R 2

2

2

3

l

MgProto 6 M E )

= H ; R = A l k . 2 _ M V . 7 A l k . E , 6 - C O O H , Mg P r o t o ( M V M g P r o t o

3

g. R = _ C H = C H ; R

4

4

3

7ester)

= H ; R = H ; D V M g Proto 4

= H; = R = H ; 2 _ M V M g Proto 4

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 . )


300


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


22.

REBEIZ ET

AL.


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 ) .



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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


22.

REBEIZ ET AL.


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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 .


306


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


22.

REBEIZ ET AL.

Photodynamic

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


308


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



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


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


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


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


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


picolinic acid


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


,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


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


22.

REBEIZ ET AL.

Photodynamic

Herbicides

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


318


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



4,4'-Dipyridyl



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


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



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


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


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


2,2'-dipyridyl


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%


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.



4,7-phenan throline



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


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


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


2,4-dipyridyl


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


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.


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.



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


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


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


332


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


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


334


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


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


336


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


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


338


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


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


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